Sequence-specific detection of nucleotide sequences

ABSTRACT

A method for detecting the presence of a target nucleotide sequence in a sample of DNA is described herein in which a test sample comprising single stranded DNA is exposed to a DNA probe and a nicking endonuclease under conditions that would permit sequence-specific hybridization of the probe to a complementary target sequence. The probe comprises a sequence complementary to the target sequence to be detected and this sequence also includes a recognition sequence for the nicking endonuclease. If the sample contains the target sequence, the probe hybridizes to the target and is cleaved by the nicking endonuclease, which leaves the target intact. Observing the presence of probe cleaved by the nicking endonuclease indicates the presence of the target nucleotide sequence in the sample of DNA.

GOVERNMENT SUPPORT

This work was supported in part by contracts W911SR-05-C-0029 and W911SR-04-C-0036 from the US Department of Defense. The U.S. government mayhave certain rights in the invention.

BACKGROUND

1. Technical Field

The present disclosure relates to methods for the detection of specificnucleotide sequences and reagents and kits for use in practicing themethods.

2. Related Art

Nicking endonuclease enzymes have been previously described. Forexample, U.S. Pat. No. 6,191,267 discloses recombinant DNA encoding anicking endonuclease, N.BstNBI, and the production of N.BstNBIrestriction endonuclease from the recombinant DNA utilizing PleImodification methylase. U.S. Pat. No. 6,395,523 discloses two methods toengineer nicking endonucleases from existing Type IIs restrictionendonucleases, and the production of engineered nicking endonucleases.One approach involves inactivating the dimerization function of a TypeIIs restriction enzyme using site-directed mutagenesis approach. Anotherapproach involves replacing the cleavage domain of a Type IIsrestriction enzyme with the cleavage domain from the naturally occurringnicking endonuclease, N.BstNBI.

SUMMARY

A method for detecting the presence of a target nucleotide sequence in asample of DNA can comprise exposing a test sample comprising singlestranded DNA to a DNA probe and a nicking endonuclease under conditionsthat would permit sequence-specific hybridization of the probe to acomplementary target sequence, wherein the probe comprises a sequencecomplementary to the target sequence that also includes a recognitionsequence for the nicking endonuclease; and, observing whether the probeis cleaved by the nicking endonuclease, wherein the presence of probecleaved by the nicking endonuclease indicates the presence of the targetnucleotide sequence in the sample DNA.

In various embodiments, the method can be multiplexed to detect thepresence of a plurality of target nucleotide sequences in a sample ofDNA. In alternative embodiments, the method can further compriseproducing amplified DNA from a biological sample. Probes can compriseboth fluorescent moieties and fluorescence quencher moieties inproximity such that while the probe remains whole, no fluorescence isobserved. When such the probes are cleaved, increased fluorescence canbe detected, permitting real-time observation of probe cleavage. Inalternatives, probes can be attached to a solid surface, for example ina micro array or on the surface of microbeads.

The technology can be applied to the detection of bacterial pathogens inenvironmental samples, for example a method of detecting Bacillusanthracis is described. Alternatively, the technology can be applied tothe detection of viral RNA in crude samples having significantbackground RNA.

These and other variations are set forth in greater detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Overview of the streaming probe technique 1.

FIG. 2 a. Amplification by PCR of 16S rRNA gene DNA from E. coli and B.subtilis genomic DNA and the design of the E. coli-specific probe E1.

FIG. 2 b. Detection of E. coli 16S Amplicon using Streaming FRET ProbeStrategy.

FIG. 3. Detection of E. coli 16S DNA in the presence of an excess ofnonspecific DNA.

FIG. 4B. Schematic representation of a streaming reaction with Nt.Alw Iadapted for CE or gel analysis.

FIG. 4 b. PAGE Gel showing the results of mixing 100 pmoles of the E1probe with 100 fmoles of either E. coli (lane 2) or B. subtilis (lane 3)oligonucleotides E1c or B1c and 50 units Nt.Alw I in 50 μl buffer.

FIG. 4C. Output of a CE instrument in which E. coli complement was mixedwith fluorescein labeled E1 probe in equimolar concentrations asindicated.

FIG. 4D. Output of a CE instrument in which E. coli or B. subtilistarget 16S rRNA DNA was mixed with 100 pmole fluoroscein-labeled E1probe as indicated in FIG. 4C.

FIG. 5. Detection of specific DNA sequences in E. coli genomic DNA. A.Analysis of the reactions using P/ACE MDQ LIF. B. The scale was expandedto more clearly show the peaks corresponding to the 5mers.

FIG. 6. Schematic of the effect of single point mismatches between probeand target on the streaming reaction.

FIG. 7. Results of a four-plex assay. Probes against the four geneticelements shown were designed to give distinguishable fluorescentproducts using CE on an ABI 3130XL.

FIG. 8. Results showing the sensitivity of a combined MDA and streamingprobe assay.

FIG. 9. Results demonstrating that cycling the temperature duringannealing, cleavage and dissociation increases the reaction rate.

FIG. 10A. Schematic representation of a streaming reaction with N.BstNBI adapted for CE or gel analysis. In this example the probe is 19nucleotides long with a fluorescein at the 5′ end. N.BstNB I cuts 5residues from the 3′ end to give a 12mer and a 7mer.

FIG. 10B. Results of a streaming assay using Nt.BstNB I.

FIG. 11. Results of a Nt.BbvC I streaming assay.

FIG. 12. Results showing that single stranded DNA target can improvesensitivity.

FIG. 13. Exemplary Linear Fluor-Quench Probe Design.

FIG. 14. Stem-Loop Fluor-Quench Probe Design.

FIG. 15. Illustrates an exemplary procedure for identifying unique probesequences.

FIG. 16. Exemplary results using stem-loop F/Q probes.

FIG. 17. Illustrates real time results using a stem-loop F/Q probe.

FIG. 18. Illustrates real time results using a linear F/Q probe.

FIG. 19A. Illustrates a comparison of linear probe F/Q assay withcapillary electrophoresis assay.

FIG. 19B. Illustrates detection of low concentrations of target usinglinear F/Q probes.

FIG. 20. Illustrates detection of 1 fmol Target using F/Q ProbePChr97.3.

FIG. 21. Illustrates detection of 0.1 fmol Target using F/Q ProbePChr97.3.

FIG. 22. Illustrates that a hairpin F/Q Probes can give very high signalto noise ratios at reduced temperatures.

FIG. 23. Illustrates Surface-Coupled Probes (sc-probes) that release afree Fluorescent Oligonucleotide.

FIG. 24. Illustrates Surface-Coupled Probes (sc-probes) where the solidsurface increases in fluorescence upon cleavage by Nt.ALWI.

FIG. 25. Illustrates sc-Probe to Bead Coupling Efficiency.

FIG. 26: Illustrates that bead bound probes allow increased CE injectiontimes increasing sensitivity.

FIG. 27. Illustrates that probes attached to beads do not require aspin-down before analysis by CE.

FIG. 28. Illustrates that soluble and sc-Probes have similar kinetics inreal-time assays.

FIG. 29. Illustrates that scProbes work well attached to multi-wellplates.

FIG. 30. Illustrates that sc-Probes on Plates Can Be Stored Desiccated.

FIG. 31. Illustrates an assay design.

FIG. 32. Illustrates that MDA can amplify DNA from environmentalcollection filters.

FIG. 33. Illustrates efficient detection of genomic DNA in crudeenvironmental samples by NESA compared with qPCR.

FIG. 34. Illustrates a probe screening.

FIG. 35. Illustrates probe activity on B. anthracis strains and onrelated species of Bacillus.

FIG. 36. Illustrates performance of a multiplex assay for B. anthracisdetection and genotyping.

FIG. 37. Illustrates that the rNESA MS2 assay is more sensitive thanrtPCR.

FIG. 38. Illustrates that rNESA but not rtPCR is resistant toenvironmental contaminants.

FIG. 39. Illustrates the dengue virus genome organization and exemplaryprobe location.

FIG. 40. Illustrates a dengue virus serotype-specific rNESA assay.

FIG. 41. Illustrates a dengue multiplex assay.

FIG. 42. Illustrates the sensitivity of the example Dengue rNESA assay.

DETAILED DESCRIPTION

We have developed a sensitive method of identifying specific single- ordouble-stranded DNA sequences and, by extension, other nucleic acidssuch as RNA that can be converted to DNA. The method involvessequence-specific hybridization of a complementary oligonucleotide probeto a target DNA. When annealed, the oligonucleotide and target create arecognition site for a strand-specific nicking restriction endonuclease.The nicking endonuclease cleaves the oligonucleotide into two pieces,but leaves the target intact. Due to the decreased size of thefragments, their affinity for target DNA is reduced and they dissociateleaving the target free to form a new complex with full-length probeoligonucleotide. The reaction contains excess oligonucleotide (on a moleto mole basis) and so hybridization, cleavage, and dissociation occurmany times. The reaction is limited only (at least theoretically) by theavailability of oligonucleotide and the stability of the enzyme. Becauseof the possibility of continuous reaction and turnover of the probe, theassay can be called a streaming probe assay and the probes referred toas streaming probes.

The reaction can be highly specific since it requires completecomplementarity between the oligonucleotide and the target at therestriction site to allow restriction cleavage and can be conducted soas to require complete or nearly complete complementarity outside of theenzyme recognition site to allow hybridization. Hybridizationtemperatures can be adjusted to allow increased or decreasedspecificity; sequences containing just one mismatch (e.g., singlenucleotide polymorphisms (SNPs)) can be distinguished if desired.

Any technique that can determine the presence of different sized orcleaved DNA can be used to measure either the rate or the end point ofthe streaming assay reaction. For example, in one embodiment, denaturingpolyacrylamide gel electrophoresis (PAGE) is a relatively simpletechnique that can be used to detect cleavage of the probeoligonucleotide. In another embodiment, the method can be madeexceptionally sensitive by using fluorescently labeled DNA together withcapillary electrophoresis (CE). In yet another embodiment, a real timemethod can be performed using an oligonucleotide comprising afluorescent group attached at one end and a quencher on the other end ofthe oligonucleotide. In this case, cleavage and dissociation of theprobe results in increased fluorescence that can be measured inreal-time using a fluorescence reader. As one example, we demonstratethat a streaming assay can be used to specifically detect the presenceof plasmids, such as Bacillus anthracis pX01 and pX02 plasmids, E. coligenomic DNA and Bacillus subtilis genomic DNA.

The method can be used whenever there is a desire to detect a specificsequence that includes a recognition sequence of a nicking restrictionendonuclease that is known or can be engineered from a type IIsrestriction endonuclease. For instance, this method is suitable fordetecting the presence of specific DNAs in a mixture (microorganismcontamination, infection, etc.); and it can be used for SNP analysis andfor genotyping. Statistically, one or more unique target sitescontaining a suitable nicking endonuclease recognition sequence can beexpected to be found in most all DNA sequences of sufficient length.

When combined with whole genome amplification (WGA), for example usingisothermal multiple displacement amplification (MDA), it is possible tospecifically detect genomic DNA amplified from about ten bacterial cellsor less. The streaming probe assay can be multiplexed allowing thedetection of multiple sequences (multiple genes in one organism orindividual genes in multiple organisms). The multiplexing system can benoncompetitive in nature, unlike multiplexing systems that usepolymerase chain reaction (PCR), and allows the generation of highthroughput quantitative data.

A method utilizing WGA and a streaming probe can have significantadvantages over current methods such as PCR. These can include thefollowing. There is no need to purify DNA before amplification.Amplified DNA is generated that can be used directly with the streamingprobe. DNA that can be used for thousands of streaming probe reactionsis generated at once. Both DNA amplification and detection methods canbe nondestructive, i.e., the same DNA can be used for multiplesequential tests including forensic tests.

The methods described herein exploit the particular properties ofnicking restriction endonuclease enzymes. When conventional restrictionendonucleases bind to their recognition sequences in DNA, they hydrolyzeboth strands of the DNA duplex at the same time. Two independenthydrolytic reactions proceed in parallel, driven by the presence of twocatalytic sites within each enzyme, one for hydrolyzing each DNA strand.That is, restriction enzymes classically recognize a double-stranded DNAbinding site and then cleave each strand of the DNA using twoindependent catalytic cleavage centers. Nicking endonucleases, on theother hand, cut only one strand. Nt.BstNB I is a naturally occurringnicking endonuclease that only cleaves one strand due to its inabilityto form dimers (4,5). The nicking endonuclease Nt.Alw I was engineeredby creating a fusion protein between the DNA binding domain of Alw I andthe cleavage/dimerization domain of Nt.BstNB I (6). Three additionalnicking endonucleases Nt.BbvC I, Nb.BbvC I and Nb.Bsm I have beencreated. The methods described herein exploit the single-strand cleavageactivities of nicking endonucleases to provide a sensitive assay fordetecting the presence of specific sequences in a DNA sample, or anysample that can be converted to DNA containing a nicking site. Inprinciple, the assay will work with any nicking endonuclease.

Several nicking endonucleases are now available from New EnglandBiolabs. For example, N.BstNB I occurs naturally and nicks by virtue ofits inability to form dimers. N.Alw I, a derivative of the restrictionenzyme Alw I, has been engineered to behave in the same way. Both nickjust outside their recognition sequences. N.BbvC IA and N.BbvC IB arealternative derivatives of the heterodimeric restriction enzyme BbvC I,each engineered to possess only one functioning catalytic site. Thesetwo enzymes nick within the recognition sequence but on oppositestrands. Nb.Bsm I cleaves only one strand of DNA on a double-strandedDNA substrate. Nicking endonucleases are as simple to use as restrictionendonucleases.

As disclosed in U.S. Pat. No. 6,395,523, it is possible to engineerknown restriction enzymes to hydrolyze only one strand of the duplex,i.e., to produce DNA molecules that are “nicked,” rather than cleaved.Therefore, it is possible to create new specificities as desired fromthe array of known enzymes and the methods described herein can begenerally applied to any sequence for which an appropriate restrictionendonuclease exists or can be engineered. The method is not limited touse of Nt.Alw I, Nt.BstNB I, Nb.Bsm I, and Nt.BbvC I, which areexemplified herein.

Thus, a method for detecting the presence of a target nucleotidesequence in a sample of DNA can comprise:

exposing a test sample comprising single stranded DNA to a DNA probe anda nicking endonuclease under conditions that would permitsequence-specific hybridization of the probe to a complementary targetsequence, wherein the probe comprises a sequence complementary to thetarget sequence that also includes a recognition sequence for thenicking endonuclease; and,

observing whether the probe is cleaved by the nicking endonuclease,wherein the presence of probe cleaved by the nicking endonucleaseindicates the presence of the target nucleotide sequence in the sampleof DNA.

The probe need not be perfectly complementary to the target, except inthe recognition sequence. Hybridization conditions can be chosen by theskilled practitioner to provide a desired degree of sequence specifichybridization. In various embodiments, one or more base mismatches canbe permitted, or perfect complementarity can be required.

FIG. 1 illustrates an exemplary scheme for carrying out the method.Sample DNA comprising a target sequence is first denatured in thepresence of a molar excess of an oligonucleotide probe (any method ofdenaturation should work, heat denaturation was used herein). This probecontains a nicking endonuclease recognition sequence (black bar) and iscomplementary to one strand of the target DNA. 2. The probe anneals tothe target reforming the nicking endonuclease site. 3. On the additionof nicking enzyme, the probe is cleaved and the reduced affinity of thetwo resulting oligonucleotides allows them to dissociate from thetarget. Fresh full-length probe hybridizes with the target and iscleaved; the process repeats. In the example shown, the oligonucleotideprobe is labeled with a fluorescent tag on the 3′ end and a fluorescencequencher on the 5′ end (alternate positions are possible as long as thefluor and quencher are separated at the end of the reaction, and theposition does not inhibit enzymatic cleavage). Cleavage of the proberesults in separation of the fluorescent tag and quencher resulting inincreased fluorescence that can be detected in real time. The use of afluorescence tag-quencher pair is not essential. Any method that canmeasure the presence of cleaved probe is sufficient. The optimumreaction temperature will vary based on the temperatures at which theoligonucleotide probe and cleavage products dissociate from the targetunder the enzymatic buffer conditions (i.e., the melting temperature(T_(m))) and the nicking endonuclease used.

Observing whether or not the probe is cleaved can be accomplished by anytechnique that can observe the presence of shortened DNA probes or thecleavage of a fluorescently labeled probe, including poly-acrylamide gelelectrophoresis (PAGE), capillary electrophoresis (CE), and fluorescenceresonance energy transfer (FRET). Of these three techniques, CE is themost sensitive. However, FRET analysis can be performed in a real-timestreaming assay. Fluorescent labels and quenchers can be placed anywherein the probe, the only constraint being that they must not inhibit thenicking endonuclease. For instance, two fluorescent labels can be usedto increase the signal strength, or probes with different spectralcharacteristics can be used in multiplexing.

There are other possible ways of detecting the fragments. Other opticaldetection methods can be used including bioluminescence andphosphorescence techniques, with or without resonance transfer (e.g.,BRET and PRET). In addition, lanthanide-based energy transfer (LRET) canbe used to observe the separation between appropriate labels. Anotherpossibility is to use Mass Spectroscopy with or without massspectroscopy tags. Another possible method is Raman Spectroscopy.Indeed, labeling of the probe with a surface enhanced Raman sphere canincrease sensitivity many fold. Another way to detect the fragmentsproduced relates to the fact that each cleavage results in a new 3′hydroxyl and a new 5′ phosphate. The increasing presence of either canbe measured and enzymatic activity calculated.

Both single and double stranded DNA can be a target for the assay.Indeed, any DNA molecule that can be made single stranded should work inprinciple. Furthermore, with some nicking endonucleases, directdetection of RNA will be possible (7). An enzyme that recognizes andcuts RNA/DNA hybrids would work as in FIG. 1 with the substitution ofDNA target with RNA target (any kind of RNA). An alternative would be toperform an initial reverse transcriptase step to produce cDNA before thestreaming reaction. Yet a third way to detect RNA would be to constructa nicking endonuclease that contains a polynucleotide binding site thatbinds to RNA/DNA hybrids. These kinds of constructs work well withrestriction endonucleases (8) so they should also work when fused withthe nicking activity of a nicking endonuclease.

Probes can be of any suitable length as can be chosen by a skilledpractitioner with consideration of several factors including thefollowing. Probes will be preferably chosen that are of sufficientlength to permit sequence specific hybridization and sufficiently shortto permit release of the cleaved probe. The skilled practitioner willrecognize that the ideal length will be a function of the meltingtemperature (T_(m)) of the full-length probe and the T_(m)'s of the twoproducts. That is, the T_(m) differential will preferably be sufficientto allow initial hybridization of the probe followed by subsequentdissociation of both probe fragments. Where convenient, theseconsiderations can be circumvented by cycling temperatures between areaction/annealing phase and a dissociation phase. In exemplaryembodiments employing temperature cycling, the reaction/annealing phasecan be conducted at about 40° C. to about 50° C., preferably about 43°C. to about 47° C., most preferably about 45° C., and the dissociationphase can be conducted at about 50° C. to about 60° C. or at most thelimit of stability for the nicking endonuclease, for example preferablyat about 53° C. to about 58° C., e.g., at about 55° C. However, ingeneral it is to be expected that assays can be designed by choosing aprobe/target/enzyme combination that permits use of a single temperatureat which the whole reaction proceeds efficiently without having to cycletemperatures. Indeed, in preferred embodiments, using nicking enzymes ator near their maximum temperature limit (e.g., 58° C. for Nt.Alw I)yields very fast and sensitive assays.

Probes can be labeled with any appropriate labels possessing detectableoptical, mass, or resonance signatures and the like for use in any ofthe techniques described herein and similar measurement methods. Inpositioning the labels, care is preferably taken to avoid labeling aprobe in any position that will substantially impair the functioning ofthe nicking enzyme.

There are currently several nicking enzymes available. For example, NewEngland Biolabs lists Nb.BbvCI, Nb.BsmI, Nb.BsrDI, Nb.BtsI, Nt.AlwI,Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, and Nt.CviPlI in their catalog.Methods of engineering additional specificities have been developed anddescribed. Indeed DNA binding domains other than those from restrictionenzymes can be used (8). A temperature resistant nicking endonucleasethat would be compatible with PCR can be engineered. One can thenperform real-time PCR with a streaming probe and the requirement for anexact sequence match when using these enzymes should reduce falsepositive rates.

The system is very amenable to multiplexing. In one embodiment, afluorescent group can be positioned relative to the restriction site indifferent probes so that different sized fluorescent oligonucleotidesare formed from different targets. Alternatively, dyes with differentspectral characteristics can be used in probes for different targets.

A streaming assay as described herein works well on DNA amplified bymultiple displacement amplification. In exemplary embodiments,amplification occurs first and then the streaming reaction is run.However, it should be relatively simple to perform the two assayssimultaneously. The probe can be modified so that it can not act as aprimer and is resistant to the 3′ to 5′ exonuclease of the Phi29polymerase. In preferable embodiments, both reactions run at the sametemperature. Conventionally, MDA is performed at 30° C. And, thestreaming assay is preferably performed at about 45 to 59° C. However,judicious modification of annealing temperatures and/or bufferconditions and use of alternative polymerase enzymes should permit useof a single temperature.

These methods can be useful in many applications, including: detectionand identification of specific organisms in anti-bioterrorism efforts,medical applications for human and animal health, strain/speciesanalysis e.g., ecological studies; molecular biology methods includingin situ creation of a signal in a semi-fixed environment such as on asurface or in a gel, creation of a large amount of a specificoligonucleotide from a larger one, sequence-specific activation—e.g.,where cleavage product(s) but not the parent (probe) is biologicallyactive, sequence-specific inhibition—where cleavage removes a biologicalactivity of the probe, detection and quantization of levels of DNA orRNA in any system including biological systems or extracts, in vitroassays and the like; and, genomic analyses including SNP/mutationanalysis/genotyping. Test samples can include environmental sources suchas air (aerosol sampling) water, soil and the like; biological sourcescan include serum, ascites fluid, cerebrospinal fluid, amniotic fluid,synovial fluid, pleural fluid, saliva, sputum, stool, urine, semen,tissue, biopsies, swabs, and the like from human and non-human sources.The methods can be used to detect sequences in RNA and the samples cancomprise RNA, for example including viruses having RNA genomes. In suchcases, the methods described herein can comprise preparing DNA from thesample by reverse transcription. The method can be used to detect a widevariety of bacteria, viruses and parasites, such as fungi, protozoa,helminthes, and the like.

Exemplary Protocols for Generating Probes to Detect a DNA Sequence.

To generate NESA probes that detect a DNA sequence, a protocol forgenerating probes can comprise the following steps.

-   -   1. Locate all nicking endonuclease (NE) sites in the sequence of        interest, which could be a relatively small piece of DNA or a        whole genome. Can restrict search to one NE such as Nt.AlwI or        search all.    -   2. Generate oligonucleotide sequences (in silico) that have the        following preferred characteristics:        -   a. Contains a specific NE site and its surrounding genomic            sequence.        -   b. NE site positioned within the oligonucleotide so that:            -   i. when hybridized to its cognate sequence 2 fragments                are generated.            -   ii. NE cutting destabilizes the hybridization complex.        -   c. Length usually between 16 and 25 bases.        -   d. Melting temperature (Tm) is close is close to the optimum            for the temperature used for the assay, e.g. for NtAlwI this            is approximately 54° C.        -   e. For linear probes, a duplex NE site will not be generated            from hairpin formation or sequence homodimerization. For            stem-loop probes, a duplex will not permit cleavage of stem            structure in absence of target sequence.        -   f. For linear probes, the sequence lacks stable predicted            structure such as hairpins, dimers etc.    -   3. Design size of cleavage products. The NESA reaction generates        oligos of defined size. Where detection of cleavage is by        observing size of cleaved probes, any method that can monitor        the generation of these different fragments can be used to        quantify the reaction.        -   Examples include but are not limited to:            -   a. Sizing using gels such as polyacrylamide gels.            -   b. Capillary electrophoresis.            -   c. Release of part of the oligo from a solid surface.            -   d. Separation of two chemical moieties due to cleavage                of the oligo as in FRET analysis.    -   4. Alternatively, design label arrangement. Although the        reaction can be followed using unlabeled oligonucleotides,        increased sensitivity can be achieved by labeling with:        -   a. One or more fluorescent groups (including fluorescent            beads) placed so that they do not inhibit the NE.        -   b. Radioactivity.        -   c. Raman spectroscopy reporters.

To generate probes that discriminate between two DNA sequences aprotocol can comprise the following steps.

-   -   1. Locate all nicking endonuclease (NE) sites in the sequences        of interest for one or more NE.    -   2. From each DNA, in silico, generate DNA fragments containing        NE sites with enough surrounding sequence to make approximately        16 base sequences. This will generate a group of sequences from        each DNA sequence of interest.    -   3. Perform pattern matching between the fragments using a        program such as BLAST.    -   4. Discard all sequences that are 100% identical and are found        in both DNA sequences.    -   5. The remaining sequences are then designed into probes of        length 16 to 25 bases as described above (steps #2 to #5)    -   6. If desired, multiple computer generated probes from each NE        site can be evaluated with respect to length, G:C content, Tm,        secondary structure and similarity to DNA from other DNA        sequences.    -   7. Pattern matching on 16 base fragments speed up the analysis.        This can be advantageous when designing a probe specific to one        genome that does not match any DNA from any other species. The        initial matching is not essential. The length of the initial        match can be altered depending on the complexity of the two        sequences being studied

Identifying unique probe sequences. FIG. 15 illustrates a flow diagramof an exemplary protocol for identifying unique sequences. Uniquesequences can be theoretically unique by virtue of lack of appearance inany other source in the GenBank database. To identify unique probesequences a skilled practitioner can use any appropriate bioinformaticssearch program to find every sequence of defined length, preferablyabout 16 to 25 nucleotides, that contains a nicking enzyme recognitionsite. Where a probe is desired to detect multiple strains of anorganism, sequences that are common or near matches among target strandscan be retained while other sequences are discarded. To identify uniquesequences, the potential probe sequences can be screened against aseries of increasingly broad panels of known sequence. For examplepotential probe targets can be screened against near phylogeneticneighbors, then class, and so on until remaining probes are screenedagainst the whole GenBank database. Screening against increasinglylarger data sets can save computational time by reducing the set ofpotential probes at each step before progressing to a screen against thewhole database. However, the sequence can be collapsed to a singlescreen against all of GenBank. At each step, near matches can be scoredin terms of mismatch location and Tm to identify potential falsepositives to be discarded.

After a set of theoretically unique probe sequences comprising a NErecognition site have been identified. Probes having desirable cleavagepatterns can be selected for validation. Probes can be validated inassays using samples comprising common contaminants e.g., bacteria,yeast, human, rodent, and vector material. Probes can be furthervalidated in assays utilizing real-world environmental samples.Validation assays can be performed using any NESA assay conditionsdescribed herein.

Theoretically unique probes which do not produce false positives invalidation assays are functionally unique.

Fluor-Quench NESA Probes

Probes comprising a fluorescent moiety and a fluorescence quenchingmoiety can provide real-time observations of probe cleavage and otheradvantages. The assay and detection can be performed in a single tube orwell of a multi-well plate. Real time assays can be less time consumingthan end-point assays and offer the possibility of rate quantificationas a function of target concentration. Assays can be performed inparallel. The methods can be less expensive. Methods can be performed ina plate reader or qPCR machine, present in most molecular biology labs.

Linear Fluor-Quench NESA Probes contain at least one fluorescent groupand at least one quencher. The fluorescent moiety and quencher can be oneither side of the cleavage site. Cleavage separates the fluorescentmoiety and quencher increasing overall fluorescence. The fluorescentmoiety and quencher can be located on the ends of the probe or can beinternally linked.

Hairpin Fluor-Quench NESA Probes contain one or more stem loops to bringthe fluorescent moiety and quencher close together. Preferably, theprobe should have lower background fluorescence relative to a linearprobe due to more efficient quenching. Two forms of stem loop arepreferred. The nuclease recognition site can be in the stem section butonly one strand comprises a complete nicking enzyme recognition sequenceso as to avoid background cleavage. The nuclease recognition site canalso be positioned in a single stranded loop.

FIG. 13 illustrates an exemplary linear fluor quench probe design. Allprobes contain a single strand specific nuclease recognition site, afluorescent residue and a quencher. The quencher reduces thefluorescence of the fluor when the two molecules are in the sameoligonucleotide. Cleavage by the endonuclease separates the fluor andquencher by cleaving the oligo between the two groups. These groups canbe placed anywhere on the molecule denoted by the dotted blue lines aslong as they do not prevent cleavage by the nuclease including betweenthe nuclease recognition and cleavage sites. The most efficientquenching would be expected using a fluor or quencher between therecognition site and the cleavage site and a cognate fluor or quenchjust the other side of the cleavage site. Any compatible fluor andquencher can be used. In general, most probes that were synthesized used6-carboxyfluoroscein (FAM) at the ends or dT-fluorescein internally, andeither a Black Hole quencher internally or Iowa Black at the ends.

FIG. 14 illustrates exemplary stem-loop fluor-quench probe designs.Illustrated probes contain a single strand specific endonuclease (NE)recognition site, a fluorescent residue and a quencher. The fluor andquencher are held close together by a hairpin. The quencher reduces thefluorescence of the fluor when the two molecules are in the sameoligonucleotide. In the presence of target, the hairpin does not formand cleavage by the endonuclease separates the fluor and quencher bycleaving the oligo between the two groups. The NE site can be located inthe hairpin (requiring a mutation in the cognate strand) (A) or in theloop (C). The fluor and quencher can be placed on either end of themolecule. The most signal to noise would be expected with a cleavagesite that releases the fluor or quencher so that a hairpin fluor-quenchpair cannot be reformed (B). In general, most probes that weresynthesized used 6-carboxyfluoroscein (FAM) as the fluorescent residue,and Iowa Black as the quencher.

Linear probes can give good signals regardless of the specificarrangement of fluor and quencher. Probes against different targets havecan be made to have similar properties. Optimal signal to noise ratiocan be obtained when the fluor and quencher were within a fewnucleotides of each other. The streaming probe cleavage reaction canoccur immediately on addition of enzyme. These probes operate over awide range of concentrations. Slightly higher sensitivity can beobserved with 1 pmol probe compared to 10 pmol.

Hairpin probes can give good signals regardless of the specificarrangement of fluor and quencher. Nicking enzyme recognition sites canbe in the stem or loop sections. Endpoint measurement of F/Q probes canincrease the signal to noise ratio dramatically.

Surface-Coupled (sc) NESA Probes

Probes coupled to a solid surface can have a number of advantages. Ifdesigned so that cleavage removes a fluorescent tag from the surface,just product can be measured by CE, decreasing background.Alternatively, a decrease in bead fluorescence can be monitored. Becausethe only fluorescence in solution is the product (signal) reactions canbe monitored in a fluorometer or qPCR machine.

Many solid surfaces can be used, eg polystyrene beads, surfaces ofplastic multi-well dishes, magnetic beads, surface-modified glassslides. Sc-probes containing a fluor and quencher have added advantages(scQ/F probes). The assay and detection can be performed in a singletube or well of a multi-well plate. Real time assays can be performedthat are less time consuming than end-point assays and offer thepossibility of quantification. Assays can be performed in parallel andare inexpensive. They can be performed in a plate reader or qPCRmachine, present in most molecular biology labs. Multiplex is possible.Assays can be set up so that the reaction increases the fluorescence ofa solid surface. The use of surface coupled probes allows the productionof microarrays.

Standard NESA Probes hybridize specifically to target and contain anicking endonuclease site such as Nt.AlwI and a fluorescent groupattached to the oligonucleotide either at one end or internally.Surface-coupled NESA Probes also have a modification that allowscoupling to a solid surface. The sc-probe can be coupled via the 5′, 3′or an internal residues to a surface as long as the molecule is stillcut by a nicking endonuclease. High density coupling can increase theeffective concentration of probe. Fluor-Quench sc-NESA Probes, inaddition to a fluor and solid surface attachment site, contain one ormore quenchers. In some cases the solid surface could act as a quenchere.g. a bead that has inherent quenching activity or a quencher bound toit.

FIG. 23 illustrates Surface-Coupled Probes (sc-probes) that release afree fluorescent Oligonucleotide. Exemplary probes contain a nickingendonuclease site (e.g. Nt.ALWI), an NH₂ group that that can be attachedto a derivatized solid surface, and a fluorescent residue that isreleased from the solid surface as part of an oligonucleotide oncleavage by the enzyme. Probes C and D contain a fluorescent group and aquencher that straddle the nicking endonuclease site. Cleavage of theseprobes not only results in release of a fluorescent oligonucleotidefragment into solution, but also results in an overall increase influorescence because quenching is relieved. Fragments that are releasedupon cleavage of a solid-bound probe are indicated in red. The locationof the fluor and quencher can be located at the ends of the probe orinternally, as indicated by the dotted blue line, as long as the groupsdo not prevent cleavage by the nicking endonuclease. In general, mostprobes that were synthesized contained an Nt.AlwI site, used6-carboxyfluooscein (FAM) as the fluorescent residue, and either a BlackHole quencher internally or Iowa Black at the ends. Probes can beattached either to polystyrene beads or to multi-well plates.

FIG. 24 illustrates Surface-Coupled Probes (sc-probes) where the solidsurface increases in fluorescence upon cleavage by Nt.ALWI. Exemplaryprobes contain a nicking endonuclease site, an NH₂ group that that canbe attached to a derivatized solid surface, and a fluorescent group anda quencher that straddle the nicking endonuclease site cleavage site.Cleavage of these probes results in release of a quencher-boundoligonucleotide fragment into solution resulting in increasedfluorescence of the solid surface. Fragments that are released uponcleavage of a sc-probe are indicated in red. The location of the fluorand quencher can be at the ends of the probe or internally, as indicatedby the dotted blue line, as long as the groups do not prevent cleavageby the nicking endonuclease. In general, most probes that weresynthesized used 6-carboxyfluoroscein (FAM) as the fluorescent residue,and either a Black Hole quencher internally or Iowa Black at the endsand contained an Nt.AlwI site.

As described and exemplified herein, a method for detecting the presenceof an RNA sequence in a sample of biological material, the methodcomprising: (a) performing a reverse transcription procedure capable ofreverse transcribing RNA into a complementary DNA nucleotide, (b)performing a whole genome amplification technique such as multipledisplacement amplification to amplify DNA in the sample of biologicalmaterial to form an amplified sample product; (c) exposing all or partof the amplified sample product to a DNA probe and a nickingendonuclease under conditions that would permit sequence-specifichybridization of the probe to a complementary target sequence, whereinthe probe comprises a sequence complementary to a unique sequence knownto be present in the transcript of the RNA sequence that also includes arecognition sequence for the nicking endonuclease; and, (d) observingwhether the probe is cleaved by the nicking endonuclease, wherein thepresence of probe cleaved by the nicking endonuclease indicates thepresence of the RNA sequence in the sample of biological material.

This method can be performed where the RNA sequence is a viral RNApathogen genome. The method can also be performed in multiplex to permitdetection of one or more different RNA and/or DNA genomes by exposingall or part of the amplified sample product to a DNA probe and a nickingendonuclease comprises simultaneously exposing all or part of theamplified sample product to a plurality of DNA probes directed to aplurality of different pathogens and/or pathogen strains, wherein eachprobe comprises a sequence complementary to a unique sequence known tobe present in the transcript of an RNA genome of a pathogen that alsoincludes a recognition sequence for the nicking endonuclease or a uniquesequence known to be present in a DNA genome of a pathogen that alsoincludes a recognition sequence for the nicking endonuclease.

As noted above, the sample of biological material can comprise aplurality of unpurified biological contaminants. For example, the samplecan be an unpurified environmental air sample washed from a collectiondevice such as an air filter or a liquid sample. The sample can beconcentrated and/or buffered, but need not be purified or filtered.

A method for detecting the presence of a target nucleotide sequence in asample of DNA can alternatively comprise (a) exposing a test samplecomprising single stranded DNA to a nicking endonuclease and a substratesurface onto which a DNA probe is affixed under conditions that wouldpermit sequence-specific hybridization of the probe to a complementarytarget sequence, wherein the probe comprises a sequence complementary tothe target sequence that also includes a recognition sequence for thenicking endonuclease; and, (b) observing whether the probe is cleaved bythe nicking endonuclease, wherein the presence of probe cleaved by thenicking endonuclease indicates the presence of the target nucleotidesequence in the sample DNA.

Such surface coupled probes provide convenient packaging, storage, anddetection. The substrate surface onto which a DNA probe can comprise thesurface of a plastic or glass bead. The substrate surface onto which aDNA probe is affixed can also comprise a surface of a well, for examplein a multiple well plate. Detection can be by observing the release of afragment of cleaved probe from the surface. In such a method, the probecan comprise a fluorescent tag that is released from the substratesurface if the probe is cleaved by the nicking endonuclease andobserving whether the probe is cleaved by the nicking endonucleasecomprises detecting the presence of fluorescent tag released from thesurface.

Prior to the assay, the substrate surface onto which the DNA probe isaffixed can be kept desiccated. Assays using surface coupled probes canbe multiplexed using a plurality of substrate surfaces, each substratesurface comprising a different DNA probe. For example a bead can containone or more probes, and one or more beads can be used in one NESAreaction. Alternatively, a multiwell plate can be constructed with oneor more probes in one or more wells. For example, sixteen wellscontaining three probes each permit simultaneous screening of 48samples.

In another aspect, a method for detecting the presence of a targetnucleotide sequence in a sample of DNA can comprise: (a) exposing a testsample comprising single stranded DNA to a DNA probe and a nickingendonuclease under conditions that would permit sequence-specifichybridization of the probe to a complementary target sequence, whereinthe probe comprises a sequence complementary to the target sequence thatalso includes a recognition sequence for the nicking endonuclease, afluorescent tag, and a fluorescence quencher, the tag and quencher beingsituated on different sides of the recognition sequence for the nickingendonuclease, a first stem portion of the probe being capable ofhybridizing to a second stem portion of the probe, the first and secondstem portions being separated by a loop portion, the tag and quencherbeing located in the probe such that the quencher is effective to quenchfluorescent emissions of the tag when the stem portions are hybridizedto each other; and, (b) observing whether the probe is cleaved by thenicking endonuclease, wherein the presence of fluorescent emissions ofthe fluorescent tag indicates the presence of the target nucleotidesequence in the sample DNA.

The probe can be designed such that the recognition sequence for thenicking endonuclease is located in the loop portion, or the recognitionsequence for the nicking endonuclease can be located in one stem portionwhere the other stem portion includes a mismatch so that the probe doesnot comprise a duplex recognition sequence for the nicking endonuclease.

A DNA probe can include a sequence complementary to a unique sequence ofa target DNA molecule that also includes a recognition sequence for anicking endonuclease, a fluorescent tag, and a fluorescence quencher,the tag and quencher being located on different sides of the recognitionsequence for the nicking endonuclease, a first stem portion of the probebeing capable of hybridizing to a second stem portion of the probeunless the probe is cleaved at a cut site of the nicking endonuclease,the first and second stem portions being separated by a loop portion,the tag and quencher being located in the probe such that the quencheris effective to quench fluorescent emissions of the tag when the stemportions are hybridized to each other. Again, the recognition sequencefor the nicking endonuclease can be located in the loop portion or therecognition sequence for the nicking endonuclease can be located in onestem portion and the other stem portion includes a mismatch so that theprobe does not comprise a duplex recognition sequence for the nickingendonuclease.

A substrate for surface coupled probes can comprise a surface onto whicha DNA probe is affixed where the probe comprises a sequencecomplementary to a unique sequence of a target molecule sequence thatincludes a recognition sequence for a nicking endonuclease. Thesubstrate can be a plastic or glass bead, or a multiwell plate where oneor more of the wells comprising one or more different DNA probes, eachdifferent probe comprising a sequence complementary to a unique sequenceof a target molecule sequence that includes a recognition sequence for anicking endonuclease. The probe can comprise a fluorescent tag that isreleased from the substrate surface if the probe is cleaved by a nickingendonuclease. The substrate surface onto which the DNA probe is affixedcan be stored desiccated, for example when beads or a mutiwell plate isa part of a kit for performing a NESA assay. A kit can comprise aplurality of substrates, e.g. plates, beads, and the like, differentsubstrates comprising one or more different probes.

The following examples serve to further illustrate various aspects andembodiments of the methods described herein. These examples should notbe considered limiting in any way.

EXAMPLES Materials and Methods

The following materials and methods are used in the examples belowunless otherwise indicated.

Genomic DNA. Genomic E. coli and B. subtilis genomic DNAs were suppliedby Molecular Staging Incorporated and were generated using MDA (9) from100 E. coli cells using their REPLI-g® kit. Real-time polymerase chainreaction (PCR) was used to assess the purity of the genomic DNAs (notshown). B. subtilis DNA was also generated in house using Qiagen'sREPLI-g® kit. Genomic DNA from all other organisms was generated usingQiagen's REPLI-g® kit. The identity of the genomic DNA was confirmed byPCR-sequencing.

PCR amplified DNA for use in streaming assay reactions. The 16 S genesfrom E. coli and B. subtilis were amplified by PCR using the primers16Sf (1) and 16Sr (2). Single stranded 16S DNA was prepared using singleprimer (16Sr) PCR and the amplicon. PCR primers were designed onsequences within the E. coli (E-oligonucleotides) or B. subtilis(B-oligonucleotides) 16S RNA genes. PCR reactions were set up using theDyNAzyme PCR reaction kit (MJ Research) with 25 pmoles primers and with30 cycles (1 min @ 94° C., 1 min @ 55° C., 1 min @ 72° C.) proceeded bya 10 min @ 95° C. denaturation step and followed by a 8 min @ 72° C.extension step.

Oligonucleotides. Oligonucleotides used in these examples are shown inTable 1. Nicking endonuclease sites are in bold, mutations from thewild-type sequence are in lower case. E probes; E. coli based; B probes,Bacillus subtilis based; c probes, complement of a probe sequence.

TABLE 1 Oligonucleotide Probes N.Alw I Probes and Complements (c) E1GT GGATC AGAATGCCA E1c TGGCATTCT GATCC AC B1 GC GGATC AGCATGCCG B1cCGGCATGCT GATCC GC B2 CC GGATC TGAGGTAACGATGT E1c m1 aGGCATTCT GATCC ACE1c m2 TcGCATTCT GATCC AC E1c m3 TGcCATTCT GATCC AC E1c m4TGGgATTCT GATCC AC E1c m5 TGGCtTTCT GATCC AC E1c m6 TGGCAaTCT GATCC ACE1c m7 TGGCATaCT GATCC AC E1c m8 TGGCATTgT GATCC AC E1c m9TGGCATTCa GATCC AC E1c m10 TGGCATTCT cATCC AC E1c m11 TGGCATTCT GtTCC ACE1c m12 TGGCATTCT GAaCC AC E1c m13 TGGCATTCT GATcC AC E1c m14TGGCATTCT GATCg AC E1c m15 TGGCATTCT GATCC tC E1c m16 TGGCATTCT GATCC AgN.BstNB I probes and Complements (c) E2 CTT GAGTC TCGTAGAGGGG E2cCCCCTCTACGA GACTC AAG B2c CTCCTCTTCTG CACTC AAG Nt.BbvC I probe BB-1AATTAT CCTCAGC GCCTTT PCR 16 S amplicon Probes 16Sf ACTCCTACGGGAGGCAGC16Sr GACGGGCGGTGTGTACAA

Nicking Endonucleases. Table 2 summarizes the nicking endonucleases usedin these examples. All nicking endonucleases were obtained from NewEngland BioLabs. Reaction conditions were as suggested by themanufacturer. The amount of enzyme, target DNA, and oligonucleotideprobe used, and the length, temperature and volume of the reaction,varied from experiment to experiment and are given in the text and/orfigure legends.

TABLE 2 Streaming Assay Nicking Enzyme Recognition Site Complement MDANt.ALW I 5 bp YES YES Nb.BSM 6 bp YES YES Nt.BbvC I 7 bp YES YES

PAGE Samples were separated on a 20% polyacrylamide, 7 M urea gel usinga standard procedure (3).

Fluorescence assays using a FRET probe. Oligonucleotides wereconstructed that had a 5′ fluorescence quencher (Iowa black, IDT) and a3′ fluorescent group, Alexa 488. Probe streaming reactions wereperformed in a multi-well plate and analyzed on a SpectraMax Gemini EM,Molecular Devices' fluorescence plate reader using an excitationwavelength of 484 nm and an emission wavelength of 525 nm. The relativefluorescence units shown represent the actual readings minus backgroundfluorescence.

Fluorescence assays using Capillary Gel electrophoresis.Oligonucleotides were constructed that had either a 5 or a 3′fluorescein group. Two instruments were used for this analysis, either aBeckman P/ACE MDQ LIF or an AB1 3130XL. Electrokinetic loading was usedin all cases. For the Beckman P/ACE MDQ LIF instrument, the distancefrom the loading point to the detector was either 20 cm or 10 cmdepending on the experiment and the eCAP ssDNA 100-R kit from Beckmanwas used with voltages between 9,000 and 30,000 volts and loading timesof 2 to 10 seconds. In analyses using the ABI 3130XL, the POP6 polymerwas used on a 36 cm capillary using ABI's Fragment Analysis Protocol.

Example 1 FRET-based Streaming Assay

In the streaming assay, as in the non-streaming assay, theoligonucleotide probe is cleaved into two shorter products. There are anumber of ways of measuring this cleavage. One way is to measure thechange in fluorescence resonance energy transfer between a donor and anacceptor fluorescent moieties, or a fluorescent moiety and a quencher,arranged on opposite side of the cleavage site on a probe, e.g., a FRETassay.

Usually, when a fluorescent molecule is activated by a certainwavelength of light it emits light (fluoresces) at a longer wavelengthand this emitted light can be measured using a fluorometer. In FRET,when an acceptor or quencher is present in close proximity to afluorescent molecule (i.e., a donor), rather than fluoresce, the energyis absorbed by the acceptor or quencher that can or can not (i.e., adark quencher) emit light at an even longer wavelength.

By arranging a fluor moiety and a quencher moiety in each of theproducts of parent probe cleavage, i.e., on opposite sides of the probecleavage site, the parent probe will be quenched due to the proximity ofthe fluor and quencher but not quenched in the cleaved probe products.That is, cleavage of the probe by a restriction endonuclease physicallyseparates the quencher from the fluor, thereby reducing quenching andcausing a measurable increase in fluorescence. This kind of assay isillustrated in FIG. 1. Because the acceptor or quencher can emit at alonger wavelength than the donor fluor, as alternatives to observing anincrease, in the fluorescence of the fluor, it is also possible toobserve a decrease in emission from an acceptor, or to observe changesin the relative intensities of emission at donor and acceptor emissionwavelengths.

Target DNA is first denatured by heating at 95° C. for 10 min in thepresence of a molar excess of an oligonucleotide probe (FIG. 1). Thetarget DNA can be any DNA that is, or can be made, single-stranded. Wehave used oligonucleotides, PCR-amplified DNA, and genomic DNA(Materials and Methods). The probe is an oligonucleotide that has aquencher on the 5′ end and a fluor on the 3′ end. Although, in thisexample quencher and fluor are on opposite ends of the probe, eitherfluor or quencher can be placed anywhere within each of the fragments aslong as they do not inhibit enzymatic cleavage and as long as they endup on different cleaved products (below). The probe also contains anicking endonuclease recognition sequence (black bar) and iscomplementary to one strand of the target DNA; the probe anneals to thetarget reforming the nicking endonuclease site. On the addition ofnicking enzyme, and incubation at a suitable temperature (discussedbelow) the probe is cleaved and the reduced affinity of the tworesulting oligonucleotides allows them to dissociate from the target.Fresh, full-length probe hybridizes with the target and is cleaved.Cleavage of the probe results in separation of the fluorescent tag andquencher resulting in increased fluorescence.

Theoretically, in the presence of active enzyme the reaction shouldrepeat continuously until near completion. The overall sensitivity ofthe assay is therefore largely a factor of how much fluorescent probe isavailable for cleavage and the background (quenched) level offluorescence.

Example 1.1 Detection of the E. coli 16S rRNA Gene in pcr-Amplified DNAUsing the Fret-Based Streaming Assay

An oligonucleotide probe was developed that hybridizes to E. coli 16SDNA and is cleaved by Nt.Alw I. This probe has reduced binding affinityfor B. subtilis 16S DNA due to 3 nucleotide differences between the 16 SDNA of the 2 species (Tablet, FIG. 2 a). FIG. 2 a shows amplification byPCR of 16S rRNA gene DNA from E. coli and B. subtilis genomic DNA andthe design of the E. coli-specific probe E1. The forward and reverseprimers are designed to amplify 16 S ribosomal DNA from many bacterialspecies including E. coli and B. subtilis. (Materials and Methods). Theprobe was designed based on the presence of a N.Alw I or N.BstNB I siteand a melting temperatures between 48 and 54° C. Red letters representthe N.Alw I and N.BstNB I recognition sites. B. subtilis sequence thatis not identical to E. coli sequence is shown in lower case blue.

The utility of this probe was tested using PCR-amplified 16S DNA from E.coli and B. subtilis. The amplification was performed using a set ofuniversal primers (FIG. 2 a).

FIG. 2 b shows the detection of E. coli 16S Amplicon using a streamingFRET probe strategy. 100 fmole (65 ng) 16 S E. coli or B. subtilis DNAwere incubated with 100 pmole 16S E. coli specific FRET probe E1,denatured at 95° C. for 10 minutes and incubated with 50 units N.AlwI at45° C. in a total volume of 200 μl. Fluorescence was determined at theindicated times.

As can be seen in FIG. 2 b, starting with 100 fmoles of E. coli 16Samplicon, a signal above background (we used a level of 3-times thestandard deviation of the background as our cut off point) can bedetected. Whereas with the B. subtilis DNA, no signal above the cut offwas detected even after 90 min. FIG. 2 b also demonstrates that thecleavage reaction and resultant increase in fluorescence can be detectedin real-time.

Example 1.2 Detection of E. coli 16S PCR-Amplified DNA in the Presenceof Excess Nonspecific DNA Using the Fret-Based Streaming Assay

FIG. 3 shows the detection of E. coli 16S DNA in the presence of anexcess of nonspecific DNA. 100 fmole (65 μg) 16S rRNA E. coli ampliconDNA together with the indicated amounts of genomic B. subtilis DNA wereincubated with 100 pmole 16S E. coli specific FRET probe E1, denaturedat 95° C. for 10 minutes and incubated with 50 units Nt.Alw I in a totalvolume of 200 μl at 45° C. Fluorescence was determined at the indicatedtimes.

Reactions were set up using the E. coli 16S amplicon and increasingamounts of 16S B. subtilis genomic (i.e., nonspecific) DNA (FIG. 3). Wefound that the addition of 5 μg of B. subtilis DNA to 65 ng of E. coliDNA resulted in little inhibition after 10 min but that this inhibitionincreased to about 25% after two hours. These results demonstrate thatspecific DNA sequences can be detected in the presence of an excess ofnonspecific DNA with only a modest decrease in efficiency. In thisdemonstration, the excess non-specific DNA is on a weight basis and nota molar ration basis. Due to the difference in target and non-specificDNA molecule sizes the molar ratio of non-specific to target moleculesis less than 1, but there is no reason to believe that the same resultswould not be obtained with a similar molar excess of molecules havingsimilar sizes.

Example 2 A Simple Denaturing-Acrylamide Gel-Based Streaming Assay

For some applications a simple, low cost assay is most appropriate. Todemonstrate such an embodiment of the method, a reaction was performedusing a probe comprising a fluorescein residue at the 5′ end and withouta quencher residue (FIG. 4B).

FIG. 4B shows a schematic representation of a streaming reaction withNt.Alw I adapted for CE or gel analysis. In this example the probe is 16nucleotides long with a fluorescein at the 3′ end. Nt.Alw 1 cuts 5residues from the 3′ end to give an 11 mer and a 5mer. The 5mer retainsthe fluorescein and can be detected using CE or PAGE (or any othertechnique that separates according to size).

FIG. 4 b shows a PAGE gel showing the results of mixing 100 pmoles ofthe E1 probe with 100 fmoles of either E. coli (lane 2) or B. subtilis(lane 3) oligonucleotides E1c or B1c and 50 units Nt.Alw I in 50 μlbuffer. After one hour samples were diluted 1:1 with loading buffer and20 μl was loaded onto a 20% polyacrylamide, 7 M urea gel. Lane 1contains marker oligonucleotides of 5, 16 and 11 residues. The gel wasvisualized on a UV light box. The 5mer fragment runs slightly fasterthan the 5mer standard because it contains a 5′ phosphate (the standardsdo not).

The 5mer probe cleavage product is clearly visible in lane 2 where thesample comprised 100 fmoles E. coli complement oligonucleotide (E1c) butnot in lane 3 where the sample comprised 100 fmoles B. subtiliscomplement oligonucleotide (B1c). The 11 mer cleavage product is notseen because it does not retain the fluorescent label.

Example 3 A Highly Sensitive Capillary Electrophoresis (CE) Assay

Capillary electrophoresis (CE) can be used to detect probe cleavage andcan provide a more sensitive alternative to the FRET assays ofExample 1. CE separates charged molecules by their size and has longbeen used to separate DNA fragments. The Beckman P/ACE MDQ LIF system isprogrammable and has the ability to detect very small oligonucleotides.However, any suitable CE system can be used. The CE assay was set up inthe same way as the denaturing acrylamide gel assay shown in FIG. 4B. Itdiffers from the FRET assay in that the probe comprises a singlefluorescence molecule at the 3′ end of the molecule. That is, a quencheris not required and the position of the single fluorescence residue canbe on the 3′ or 5′ ends, or internal as long as it does not inhibitendonucleolytic cleavage. Cleavage of the 16mer probe results inproduction of a fluorescently labeled 5mer that can be detected by CEand an unlabelled 11mer that is not observed.

A CE-based assay was also used to examine the effects of varying thelevels of target and probe. FIG. 4C shows the output of a CE instrumentin which E. coli complement was mixed with fluorescein labeled E1 probein equimolar concentrations as indicated. Samples were denatured for 10min at 95° C. and cooled to 45° C. 50 units Nt.Alw I were added and thereaction cycled between 45° C. (1 min) and 55° C. (10 sec) for 2 hours.Samples were diluted 1000-fold and electrokinetically (5 s) loaded ontothe capillary of a Beckman P/ACE and run at 9,000 volts for the timeindicated. The detector was 20 cm from the loading end of the capillary.The doublet seen at the position of the 5mer is most likely a loadingartifact.

The 5mer product eluted at about 18.4 minutes and was only seen when E.coli 16S amplicon was present. At levels of 100 and 10 pmoles of targetand probe, nearly all the E1 probe was cleaved. As levels were reducedfurther there was a reduced signal but still clearly visible signal withjust 100 fmoles of target and probe. The initial reaction was performedin 200 μl, and a 1 μl sample of this reaction was diluted into 100 μlbefore electrokinetic injection. It is expected that only a fraction ofthe material present in the sample enters the capillary tube. Thus the5mer peak seen with the original 100 fmoles probe and target reaction,reflects the signal obtained from far less than 500 attomoles target.This shows a remarkable sensitivity especially given that the probe andtarget were in equimolar amounts rather than the probe being in excess.We also determined the effects of decreasing the amount of target whileretaining a constant level of probe (100 pmoles). As can be seen in FIG.4D, the reaction is specific. No signal is generated by 1 pmole B.subtilis amplicon DNA. However, as little as 10 fmoles of E. coli 16 Samplicon gave a positive signal.

In FIG. 4C, the 5mer elutes at approximately 18.3 minutes. To increasethe speed of separation we used a 10 cm load to read setting (previouslywe had used 20 cm) and we increased the voltage to 30,000 from 9,000volts. Using this set up the 5mer eluted at approximately 2.9 minutes.FIG. 4D shows the output of a CE instrument in which E. coli or B.subtilis target 16S rRNA DNA was mixed with 100 pmolefluoroscein-labeled E1 probe as indicated in FIG. 4C. Samples weredenatured for 10 min at 95° C. and cooled to 45° C. Fifty units ofNt.Alw I were added (total volume 200 μl) and the reaction cycledbetween 45° C. (1 min) and 55° C. (10 S) for 2 hours. Samples wereelectrokinetically (5 sec) loaded onto the capillary of a Beckman P/ACEand run at 30,000 volts for the time indicated. The detector was 10 cmfrom the loading end of the capillary.

Example 3.1 Detection of E. coli Genomic DNA (MDA) Using the CE-BasedStreaming Assay

The sensitivity of the assay demonstrated in FIG. 4, indicates that theassay can be sensitive enough to detect short specific sequences withingenomic DNA. To demonstrate this, we performed the assay using a Nt.AlwI probe on E. coli genomic DNA (Materials and Methods).

FIG. 5 shows the detection of specific DNA sequences in E. coli genomicDNA. Genomic DNA (0.25 μg/μl was denatured at 95° C. for 10 min in thepresence of probe E1 (100 pmole). Nt.Alw 1 (50 units) was added (totalvolume, 200 μl) and the reaction cycled between 45° C. (1 min) and 55°C. (10 sec) for the indicated time. A. Analysis of the reactions usingP/ACE MDQ LIF. Samples were diluted 1000-fold and 100 μl (25 ng genomicDNA) were subjected to a 5 sec electrokinetic loading and run at 30,000volts. B. The scale was expanded to more clearly show the peakscorresponding to the 5mers. The position of 5 mer elution changessomewhat with repetitive CE runs due to the high voltages used. As canbe seen in FIG. 5, a positive signal (the 5mer eluting at about 2.4minutes) can be detected after a 30 min reaction. The probe used gave apositive signal with E. coli DNA and did not give a signal with B.subtilis DNA even after 120 min. This demonstrates that the streamingassay can be used to determine the presence of DNA from specificorganisms.

Example 3.2 Detection of Point Mutations Using a CE-Based Assay

The streaming assay can be used to distinguish between closely relatedDNA sequences as shown in FIG. 5. To address whether it can also be usedto detect single base pair differences, we designed primers identical tothe E1 complement used before but introduced a point mutation inindividual oligonucleotides at each position. These oligonucleotideswere then used in a streaming reaction with the E1 probe and theirefficacy determined. FIG. 6 shows the effect of single point mismatchesbetween probe and target on the streaming reaction. Streaming reactionswere set up using the E. coli E1 probe and targets that consisted of theperfect complement (E1c) oligonucleotide, complementary oligonucleotideseach with one mismatch (m1 to m16), and a B. subtilis complement thathas 3 mismatches (all outside of the N.Alw I binding site). Reactionswere performed for 2 h and the products separated by either CE ordenaturing PAGE. Black bar, no detectable cutting; vertical hash barclearly observable product; horizontal hash bar, very low activity (theboundaries were taken as the highest temperature where a reaction wasseen to occur); nucleotides in bold, Nt.Alw I site.

As can be seen in FIG. 6, a single missense mutation can be sufficientto substantially decrease the signal. Those oligonucleotides havingmutations in the recognition sequence of the enzyme were completelyinactive. Thus a streaming probe can be used to measure the presence ofsingle nucleotide mutations/polymorphisms with a properly designedprobe.

Example 3.3 Development of A Multiplex Assay Using the ABI 3130XL

CE instruments capable of separating oligonucleotides that differ insize by one nucleotide are available. Such instruments, for example theBeckman P/ACE, can be used to perform a multiplex assay where probesthat yield different lengths of cleavage product are used againstmultiple targets in one reaction. In such a multiplex reaction, eachprobe can be designed such that the cleavage site for the nickingendonuclease produces a unique sized cleavage product.

The Beckman P/ACE, is a single capillary, one-color machine so itsmultiplex ability is limited to distinguishing sizes. Anotherinstrument, the ABI 3130XL, has capacity for 16 capillaries and canhandle fluors of four different colors. With such a device, multiplexingassays can include probes that are labeled with different coloredfluorescent moieties.

The results of a four-plex assay are shown in FIG. 7. In this assay,four different probes were used in one reaction. These probes werespecific for the Bacillus anthracis plasmids pX01 and pX02 and forBacillus subtilis and were designed to yield different sized cleavageproducts when cut by a nicking endonuclease upon hybridization. Thereaction (10 μl) contained 1 pmole of each probe, 100 fmoles of eachcomplement oligonucleotide, 10 U Nt.Alw 1 and 1×NEB buffer 2. Thereaction was run for 1 hr. at 58° C. before analysis. The green peakslabeled in red are size standards. The B. subtilis probe (B2) has afluorescein at the 5′ position and gives an 11-base fragment; pX02-1 (B.anthracis pX02 plasmid probe) has a 3′ fluorescein and yields an 10-basefragment; pX01 (B. anthracis pX01 plasmid probe) has a 5′ fluoresceinand yields an 10-base fragment; pX01 2-2 (B. anthracis pX01 plasmidprobe) has a 5′ fluorescein and yields a 11-base fragment.

In the presence of the four target DNAs, four distinct signals weregenerated. These data demonstrate the capability of multiplexing. Sincethe instrument is capable of using fluors with four different colors, amultiplex assay of 16 probes is an obvious extension. Indeed, theresolving power of the capillaries is such that multiplex assays withmore than 16 probes are possible (we estimate, based on the resolvingpower of the capillaries, that a 40-plex is possible). These data, takentogether with the above examples, also show the generality of thestreaming probe, because the feasibility of the assay with threedifferent organisms (including E. coli) using both chromosomal andplasmid sequences has been demonstrated.

Example 4 Use of a Combination of MDA and Streaming Probe to DetectApproximately 10 Bacteria

One of the goals in applications such as detection of bio-warfareagents, is to detect vanishingly small numbers of organisms. Todemonstrate that the combination of MDA and streaming probe can besensitive enough to detect low levels of bacteria, a serial dilution ofa culture of B. subtilis was performed and the samples split into two.One half was used to quantitate the number of bacteria present; theother half was used to perform MDA.

FIG. 8 shows the sensitivity of the combined MDA and streaming probeassay. Log phase B. subtilis were serially diluted and each dilution wassplit into two. Half the dilution was used for a plating assay, while 1μl of the other half was used for a MDA reaction (50 μl) using aREPLI-g® kit (Material and Methods). 100 ng of the amplified DNA wasthen used for each 10 μl streaming reaction (100 ng MDA DNA, 1 pmoleprobe, 10 U Nt.Alw 158° C.). Probe 1 is B1 and probe 2 is B2 (Table 1).

As can be seen in FIG. 8, approximately 10 B. subtilis cells can bedetected by this approach. Remarkably, the MDA reaction made enough DNAfor at least 500 hundred independent streaming reactions each of whichcan be multiplexed if desired. These experiments show the feasibility ofusing this approach to detect low levels of bacteria, they also showthat crude DNA produced directly from bacteria can be used for thestreaming reaction.

Example 5 Adjustments in the Parameters of the Reaction

The use of temperature cycling: One possible rate limiting effect is thedissociation of the probe fragments from the target after the probe hasundergone endonucleolytic cleavage. Indeed, as the concentration ofcleaved probe increases with time, there could be a significantinhibition of the process. Initially our assays were set up at 45° C.However, cycling between two temperatures, a reaction temperature (45°C.) and a dissociation temperature 55° C. might lead to an increasedrate of reaction. The results of this strategy are shown in FIG. 9. 100fmole (65 μg) 16S E. coli or B. subtilis amplicon DNA were incubatedwith 100 pmole 16S E. coli specific FRET probe E1, denatured at 95° C.for 10 minutes and incubated with 50 units Nt.Alw 1. At a constant 45°C. or cycled between 45° C. (1 min) and 55° C. for 10 sec. Fluorescencewas determined at the indicated times. These values were arrived atempirically. Optimal temperatures can be determined for anyprobe/target/enzyme combination. The results show that temperaturecycling does increase the initial rate of the reaction. Nt.AIw I isstable up to at least 58° C. and at this temperature the reaction isvery efficient. Temperature cycling can be of use for enzymes whosedenaturation temperature is below the optimum reaction/disassociationtemperature.

Use of excess probe: Another possible rate-limiting step is theannealing of the probe to the target. This can be mitigated by usinghigh concentrations of probe (pmoles) to drive the reaction forward.Interestingly, as much as practically all of the probe can be seen to becut in assays containing as little as 1 fmole target, indicating a probeturnover of at least 1000.

Use of other nicking endonucleases: To demonstrate the generality of themethod using other nicking endonucleases, a probe, E2 (FIGS. 2 a, 10 a)was designed to recognize the E. coli amplicon and to be cleaved byNt.BstNB I at its recognition site. FIG. 10A shows a schematicrepresentation of the streaming reaction with N.BstNB I adapted for CEor gel analysis. In this example the probe is 19 nucleotides long with afluorescein at the 5′ end. N.BstNB I cuts 5 residues from the 3′ end togive a 12mer and a 7mer. A mismatch in the N.BstNB I recognition sitebetween E. coli and B. subtilis 16 S DNA prevents cleavage of the probein association with B. subtilis DNA. Thus, the probe can be used toidentify the E. coli target in a B. subtilis background. Detection of E.coli was tested and the results depicted in FIG. 10B. In a 200 μlreaction, 100 pmoles probe E2 were incubated with the indicatedcomplement B2c or E2c, 100 U Nt.BstNB 1 for 2 h cycling between 45° C.for 1 minute and 55° C. for 10 sec. The sample was separated by CE witha 5 sec electrokinetic injection. The full length probe is 19nucleotides long and is cleaved into a 12mer and a 7mer. Both the 19merand the 12mer are seen (the 5mer is not fluorescently labeled) in thereaction containing E. coli complement DNA (E2c) but not in controlreactions lacking any target or a reaction containing B. subtiliscomplement DNA (B2c). Similar results were obtained with the enzymeNt.BbvCI (FIG. 11 and Table 2). 1 μg E. coli MDA was incubated with 10pmole Nt.BbvC I probe BB-1 with a fluorescein label on the 5′ end (Table1), 10 U NT.BbvC I, in a final volume of 10 μl for 3 hrs at 54° C. Thereaction was analyzed on a Beckman P/ACE.

These data show that the assay is not dependent on one nickingendonuclease but that other nicking endonucleases can be used so long asthey cleave just one DNA strand.

Detection of single strand targets: One-sided PCR was used to create asingle-stranded E. coli 16 S DNA. 100 fmole (65 ng) 16 S E. coli or B.subtilis single-stranded DNA was incubated with 100 pmole 16 S E. colispecific FRET probe E1, denatured at 95° C. for 10 minutes and incubatedwith 50 units N.Alw 1 in a total volume of 200 μl. and cycled between45° C. (1 min) and 55° C. for 10 sec. At the indicated times,fluorescence was determined. Inset: 100 pmole probe was incubated with50 units of E. coli DNAse I at 37° C. Fluorescence was determined at theindicated times. As can be seen in FIG. 12, single-strand DNA worksexceptionally well in the assay. To determine the maximum fluorescencepossible in the assay, the reaction was treated with DNAse I andfluorescence determined over time. DNAse I cleaves all the DNA andshould thus give the maximum signal possible in the reaction. The datashows that in this reaction Nt.Alw I reached a remarkable level of 45%of maximum possible fluorescence.

Example 6 Fluor/Quench Probes

TABLE 2 Exemplary Fluor/Quench Probes Name Sequence (5′ to 3′) TypeSet 1 P36.1 FAM-CG C  GGATC  TTAA | Hairpin. GGCTACGTCTT GAACC GCG-IBRecognition site & point mutation on stem, 5′F, 3′Q P36.2 FAM-CGCGTT C GGATC TTAA | Hairpin. GGCTACTTAAC GCG-IB Recognition site inloop, 5′F, 3′Q P36.7 FAM-C C  GGATC  TTAA | Hairpin. GGCTACGTCTTRecognition site in AAACCTTAATTACCGG-IB stem, 5′F, 3′Q, triple mutationSet 2 P97.3 GCTAACTTGC GGATC T-F TA A | GG- Linear, F between IBrecognition & cut site, Q 3′ Set 3 D2.1 TT GGATC AT-F AG | GGTA TLinear, F between TGGATCTA-IB recognition & cut site, Q 3′ D2.2IB-TT GGATC AT AG | GGTA T-F Linear, Q 5′, F TGGATCTA internal D2.3FAM-TT GGATC AT AG | GGTA T Linear, F 5′, Q, 3′ TGGATCTA-IB Set 4 D4.1AGTGCT-F G GGATC TCAG | Linear, internal F, Q GAAGGA-IB 3′ D4.2FAM-AGTGCT G GGATC TCAG | Linear, F 5′, Q 3′ GAAGGA-IB

F, FAM; IB, iowa black; |, cut site; blue text, recognition site; redtext, fluorescent attachment site; F, fluorescein; FAM,6-carboxy-fluoroscein. All sequences written 5′ to 3′. The targetssequences are underlined in set 1, and are the complement of the probesin sets 2, 3 and 4 with the exception of the T attached to thefluorescein

Standard 20 μl NESA reactions were set up in 96-well qPCR platescontaining 10 pmol probe, 200 fmol target oligo. Reactions wereincubated at 58° C. for 5′, 2 μl Nt.AlwI added, and fluorescencemeasured every minute at 58° C. Results are illustrated in FIG. 16.

Real time measurements were taken. Standard 20 μl NESA reactions wereset up in 96-well qPCR plates containing 10 pmol probe, 200 fmol targetoligo, 2 μl Nt.AlwI. Reactions were incubated at 58° C. and fluorescencemeasured every minute. Results are illustrated in FIG. 17. Standard 20μl NESA reactions were set up in 96-well qPCR plates containing 10 pmolprobe, 1 pmol target oligo, 2 μl Nt.AlwI. Reactions were incubated at58° C. and fluorescence measured every minute.

D2.1=Den2-3′BHQ D2.2=Den2-5′BHQ D2.3=Den2-Opp D4.1=Den4-3′BHQD4.2=Den4-Opp

Results are illustrated in FIG. 18.

A linear F/Q probe nicking enzyme streaming assay was compared with anassay employing capillary electrophoresis. For F/Q assay, standard 20 μlNESA reactions were set up in 96-well qPCR plates containing 10 pmolprobe. Reactions were incubated at 58° C. for 5′, 2 μl Nt.AlwI added,and fluorescence measured every minute at 58° C. For the CE assay,standard 10 μl NESA reactions were set up with 1 pmol probe. Reactionswere incubated at 58° C. and analyzed by CE. Results are illustrated inFIG. 19.

FIG. 20 illustrates detection of low concentrations of target usinglinear F/Q probes. Lowering concentration of the probe can increasesignal. FIG. 21 illustrates detection of 0.1 fmol Target using F/Q ProbePChr97.3.

Hairpin F/Q Probes can give very high signal to noise ratios at reducedtemperatures. A 20 μl NESA reaction was set up using standardconditions. After 5 min @ 58° C., 2 μl Nt.AlwI was added and thereaction continued at 58° C. The enzyme was heat killed and cooled to25° C. For the Melt curve, the temperature was then increased 1° C.every 10 sec. Superb signal to background is obtained below 40° C.(background is virtually zero) Results are illustrated in FIG. 22.

Example 7 Surface Coupled Probes

Coupling of 3′-amino modified oligos to Qiagen LiquiChip carboxylatedmicrobeads. A 1 ml suspension of LiquiChip beads was vortexed for 2 min.Aliquots of 125 μl were transferred to microfuge tubes, centrifuged for3 min at 10,000 g, and the supernatant discarded. The pellet beads wereresuspended in 50 μl of 0.1 M MES pH 4.5, and vortexed for 30 sec. Onenmol (10 μl of 100 pmol/μl solution) of 3′-Amino modified oligospreviously resuspended in 0.1 M MES pH 4.5 were added and vortexed for30 sec. 10 μl of freshly prepared EDC([N-(dimethylaminopropyl)-N′-ethylcarbodiimide] Fluka, St Louis, Mo.)were added and the mixture vortexed for 10 sec. Coupling reactions wereplaced in a light-tight box and agitated every 30 min for 2 hours at 24°C. Coupling reactions were then centrifuged for 3 min at 10,000 g andthe supernatant discarded. 1 ml of the wash buffer (PBS pH 7.2, 0.02%Tween-20) was added to each reaction. Pellets were resuspended using apipette and then centrifuged for 3 min at 10,000 g. The supernatant wasremoved with a pipette and discarded. The washing step was repeatedtwice. A final rinse of the washed pellet was performed by the additionof 150 μl TE pH 8.0, centrifugation for 3 min at 10,000 g, and removalof the supernatant. The TE equilibrated/washed coupled beads wereresuspended in 50 μl TE pH 8.0. These surface-coupled probes (sc-probes)were stored up to four months at 4° C.

Coupling of 3′-amino modified oligos to Maleic Anhydride CoatedPolystyrene Plates. Ten μl (100 pmol/μl) of 3′-Amino modified oligospreviously resuspended in 0.1 M MES pH 4.5) were added to each well of areacti-bind amine-binding, maleic anhydride activated plate (clear,8-well strips) (Thermo Fisher Scientific Inc. Waltham, Mass.). Forty μlof PBS pH 7.4 were added to each well, and mixed by gently pipetting.Wells were sealed with OptiClear (B) film (Biorad, Hercules, Calif.) andincubated in a light tight box for 4 hours at 24° C. Plates wereagitated gently every 30 min during incubation. After 4 hours, thesupernatants were discarded, and 250 μl of blocking buffer (TE, pH 8.0)were added to each well. Plates were sealed and incubated in the darkwith occasional agitation for 1 hour. Following blocking, thesupernatants were discarded and the wells washed twice with 250 μl ofwash buffer (PBS with 0.05% Tween-20, pH 7.2). Plates were washed twiceagain with 250 μl of blocking buffer, discarding the buffer each time.To each well, 100 μl of blocking buffer were added and the platessealed. Plates were stored at 4° C. in the dark. One set of plates wasused to test if they could be stored dry. Buffer was completely removedand the plates stored at 4° C. for 48 hours.

Quantifying coupling efficiency of bead coupled probes. To determine theconcentration of the beads only, the bead-bound probes were vortexed for5 sec and diluted 100-fold using MES pH 4.5 and their concentrationdetermined using a hemocytometer. Next, 3 μl of undiluted resuspendedprobe-beads were added to 7 μl containing 1 μl of 10× DNase I buffer(Promega, Madison, Wis.), 1 μl of DNase 1 (Promega), 5 μl of H2O andincubated at 37° C. for 10 min. 1 μl of stop solution (20 mM EGTA pH8.0) was added and the reaction incubated at 65° C. for 10 min.Following this DNAse inactivation step, 19 μl of H2O were added to thereaction. Uncoupled beads, and free probe were also DNase treated foruse as background controls. Fluorescence was measured using afluorescent plate reader (excitation 495 nm, emission 520 nm). Samplefluorescence was plotted against a standard curve prepared from serialdilutions of DNAse-treated uncoupled probe. The number of fluorescentoligonucleotides attached to each bead was then calculated for eachcoupling reaction.

NESA reactions using surface coupled probes (sc-probes). Each NESAreaction contained 1 μl (1 pmol to 10 amol) of target oligonucleotide(the complementary sequence of the probe), 1 μl Restriction EndonucleaseBuffer-2 (NEB), 3 μl sc-probes, and 4 μl H2O. The 9-μl mixture washeated to 95° C. for 10 min, 4° C. for 10 sec, then 58° C. for 5 min.Once the reaction had reached 58° C., 1 μl Nt.AlwI (NEB, Ipswich, Mass.)was added followed by incubation at 58° C. for 60 min and then 80° C.for 20 min to stop the reaction.

NESA reactions using sc-probes on plates. Target oligonucleotides (thecomplementary sequences of the probes) were first denatured for 2 min at95° C. (5 μl complement (1 pmol/μl), 2.5 μl of 10× NEBuffer-2 (NEB), and17.5 μl of H2O). Each reaction was added to a well of a plate preheatedto 58° C., the plate sealed, and incubated for 5 min at 58° C. 25 μl ofpreheated diluted Nt.AlwI (2.5 μl 2.5 μl 10× NEBuffer-2 (NEB), 17.5 μlH2O) were then added and incubation continued at 58° C. for 60 min. Thereaction was then inactivated by incubating at 80° C. for 20 min. Two μlof each reaction were prepared for CE analysis as described.

Capillary gel electrophoresis (CE). Samples were analyzed using anApplied Biosystems 3130×1 Genetic Analyzer with electrokinetic injectionas described previously (6). In brief, NESA reactions centrifuged at2000 g for 5 min at 24° C. Two μl of the supernatant were diluted100-fold (20-fold with water and then 5-fold with formamide and then 10μl were used for injection. A set of Hex™-labeled standards (5, 17, 21,30, and 50 nucleotides in length) was run with each sample to aid inpeak identification.

Real time NESA (rtNESA) using sc-probes. A 10-μl reaction mixturecontaining 1 μl of diluted complement (1 pmol to 10 amol), 1 μlNEBuffer-2, 3 μl sc-probes, 5 μl H2O was heated to 95° C. for 10 min, 4°C. for 10 sec, then 58° C. for 5 min. Ten μl of diluted Nt.AlwI (2 μlNt.AlwI, 1 μl of NEBuffer-2, 7 μl of H2O) were added and the mixtureincubated for a further 60 min at 58° C. Fluorescence was determinedevery minute of the 60 min incubation using 5 s reads andexcitation/emission wavelengths of 495 nm/520 nm. All rtNESA experimentswere run and analyzed using the BioRad iQ5 RT-PCR Detection System. Theefficiency of coupling probes to polystyrene beads (4 μm diameter) wasinvestigated. On average, 0.6×106 probes were coupled to each bead. Eachbead is supposed to have 108 binding sites, approx 0.5% couplingefficiency. There can be about 3,000 beads per μl. 3 μl sc-probes usedper reaction=1.8×109 probe molecules per reaction (˜3 fmol)

For capillary electrophoresis the limit of detection can be between 100and 10 amol (10-17 M) for sc-Probes on polystyrene beads. Samples can beinjected for at least 360 s with little increase in background. Samplescan be used directly, bead removal is not necessary.

For F/Q sc-Probes on polystyrene beads, real time assays can performwell. Kinetics of sc-probes and uncoupled probes are very similar. Therecan generally be no increase in background fluorescence during thereaction. for sc-Probes on multi-well plates, very strong signals couldbe measured by CE. Sc-probe plates are stable desiccated for at least 48hours and work at least as well as “wet” plates. Desiccated plates arelikely a way for long term storage of sc-probes. Results are illustratedin FIG. 25.

TABLE 3 SC probes Probe Sequence (5′ to 3′) 1F-A GGATC TTAC GA AA CTT CGG-AmM 1c3F-A GGATC TTAC GA AA CTT CGG-(T12)-AmM 1c12AmM(C12)A GGATC TTAC GA AA CTT C-F 1.1IB-A GGATC TTAC GAT-FAA CTT CGG-AmM 1.2IB-A GGATC TTAC GA AAT-FCTT CGG-AmM 1.3IB-A GGATC TTAC GA AA CTTT-FCGG-AmM 1.12F-A GGATC TTAC GA AA CTT CGG-BHQ-AmM 1.13AmM(C6)-BHQ-A GGATC TTAC GA AA CTT CGGT-F

These probes have the same base sequence and have been aligned withspaces where necessary. F, FAM; IB, iowa black; AmM, amide linkage, BHQ,black hole quencher 1. Data using the probes in bold are shown in theseexamples. The other probes gave similar results (data not shown).

FIG. 26: Illustrates that bead bound probes allow increased CE injectiontimes increasing sensitivity. FIG. 27 illustrates that probes attachedto beads do not require a spin-down before analysis by CE.

FIG. 28 illustrates that soluble and sc-Probes have similar kinetics inreal-time assays. rtNESA Analysis of probes alone versus sc-probes.Briefly, each reaction was done in duplicate+/−1 pmol of compliment.Probe alone reactions contained 2 pmol of probe, sc-probe reactionscontained 3 ul of bead-coupled probes. (A). Real time results for probesalone, green—probe 1.1, blue—probe 1.2, red—probe 1c3. Three lines at ornear the ˜100 rfu mark represent no template control reactions,red—probe 1.1, purple—probe 1.2, brown—probe 1c3. (B). Real time resultsfor sc-probes, Green—probe 1.1, light blue—probe 1.2, yellow—probe 1c3.Three lines at or near the ˜100 rfu mark represent no template controlreactions. pink—probe 1.1, blue—probe 1.2, dark green—probe 1C3.

FIG. 29 illustrates that scProbes work well attached to multi-wellplates. FIG. 30 illustrates that sc-Probes on Plates Can Be StoredDesiccated.

Example 8 Specific Detection of B. anthracis with a Novel MultiplexAssay Suitable for Crude Environmental Samples Using Whole GenomeAmplification and Nicking Endonuclease Signal

(References cited in this example are numbered in accordance with thelisting at the end of this example.)

Introduction Although culture methods have been used for many years toidentify microorganisms, they are not so helpful with very slow growingorganisms, with those that are difficult or impossible to grow in vitro,and those organisms that do not survive harvesting methods such asfiltration. For these reasons, molecular biological methods have beendeveloped that allow the rapid identification of bacteria (7, 10, 16,17, 19). The leading technique for this is undoubtedly the polymerasechain reaction (PCR), especially quantitative PCR (qPCR), that can notonly identify an organism but can also determine relative bacterial load(15, 22). Although PCR is exquisitely sensitive and can be multiplexed,albeit with a usual decreased sensitivity, there is a caveat to usingPCR as a detection method. PCR detection from complex samples, such asenvironmental or clinical samples, often requires purification of theDNA to remove inhibitors of the PCR reaction such as heme, humic acids,chelating agents, and metals (3, 4, 12, 21). These inhibitors can giverise to false negatives that could be devastating in the case of medicaldiagnostics and catastrophic in the case of environmental surveillancefor biowarfare agents. Unfortunately the isolation and purificationprocesses necessary to obtain pure DNA are often time consuming anddifficult with field studies, while the efficiency of recovery from suchmethods can vary and affect the sensitivity of PCR detection (18). Wehave recently described a technique called nicking endonuclease signalamplification (NESA) that uses fluorescent oligonucleotide probes inisothermic cycles of hybridization, cleavage, and dissociation to detectthe presence of low levels of specific genomic DNA (5). In this report,we show that a combination of multiple displacement amplification andNESA can be used to detect genomic DNA in crude environmental sampleswithout the need for DNA with samples that are refractory to PCR. Weshow the general utility of the system by developing NESA probesspecific for B. anthracis chromosomal DNA as well as the B. anthracisplasmids pX01 and pX02 that are required for virulence. These probeswere combined in a multiplex assay that distinguishes between strains ofB. anthracis.

Materials and Methods Oligonucleotides, Genomic DNA, EnvironmentalSamples. Oligonucleotides were obtained from Integrated DNA Technologies(Coralville, Iowa). All probes were fluorescently labeled at the 5′position with 6-carboxyfluorescein (FAM™) and were purified by ionexchange HPLC. Five oligonucleotide size standards (5, 17, 20, 30 and 50bases) were synthesized with hexachlorofluorescein (HEX™) at their 5′ends. Genomic DNAs were obtained from Zyagen, San Diego, Calif. (Bovine,Cat, Dog, Monkey, Mouse, Sheep, Porcine, Equine, Human); LawrenceLivermore National Laboratory (Chicken, Drosophila melanogaster, Rabbit,Rat, Flea, Mosquito); Johns Hopkins Bloomberg School of Public Health(Tick, Ixodes scapularis); BEI, Manassas, Va., (B. anthracis strainsAmes BEI 02005259004, Vollum BEI D2005276004, GT68 BEI D2005255003, ΔSterne BEI D2004322001, GT41 BEI D2004056001, BACI055 BEI D2005027001,GT28 BEI D2006030002A1, Sterne BEI D2005075001, GT3 BEI D2004050007;Yersinia pestis strains F361/66 South America 1966 BEI 02005041002,India 1898 BEI D2005056003, 342 15-91 Russia 1960 BEI D2005060001, 338ZE942122 Zimbabwe 1994 BEI D2005042001, 346 16-34 Vietnam 1970 BEID2005056002); The Naval Medical Research Center, Silver Spring, Md. (B.anthracis Ames); ATCC, Manassas, Va. (Mycobacterium smegmatis,Thermotoga maritima, Borrelia burgdorferi, Chlorobium tepidum,Bacteroides fragilis, Shewanella oneidensis, Pseudomonas aeruginosa,Staphylococcus lugdunesis, Staphylococcus haemolyticus, Staphylococcusepidermidis, Staphylococcus saprophyticus, Staphylococcus schleiferi,Bacillus thuringiensis, Methanosarcina mazei, Spiroplasma citri,Sulfolobus solfataricus, Lactobacillus plantarum, Pyrococcus furiosus,Streptomyces avidinii, Yersinia enterocolitica, Neisseria meningitidis,Paenibacillus sp., Helicobacter pylori, Deinococcus radiourans,Clostridium aceto-butylicum, Bacillus cereus, Rhodobacter Saccharomycescerevisiae, Candida albicans); U.S. Army Edgewood Chemical BiologicalCenter (ECBC), Aberdeen Proving Ground, Md. (B. anthracis strains. Ames,ANR1, NNR-.1, NNR1-.1, VNR1-.1, Sterne; Yersinia pestis strains 9108101,A1122, Amal, 9800419, 9808723, Harbin; Staphylococcus aureus enterotoxinB positive strains ATCC 13566, 14458, 19095, 27664, 51651, 51811, 51811;Bacillus cereus ATCC 14579, Bacillus subtilis ATCC 27370, Bacillusthuringiensis BGSC 4AZ1, Bacillus thuringiensis BGSC 4G1); MolecularStaging Inc. (E. coli, B. subtilis). Genomic DNAs from pathogenicorganisms were guaranteed not to contain infectious agents by thesuppliers and licenses/permits were obtained from the appropriateagencies for purchasing and shipping. The Georgetown University'sInstitutional Biosafety Committee approved use of the genomic DNAs.BioWatch filter pieces were obtained from Lawrence Livermore NationalLaboratory. Crude extracts of these environmental filters were made byplacing the filters in 2 ml screw cap vials along with 300 μl each of 2types of acid-washed glass beads, particle size=106 μm (Sigma G4649) and425-600 μm (Sigma G8772), and 600 μl TE pH 8.0. Tubes were shaken in amini bead beater (Biospec Products, Inc #3110BX) at 4800 rpm for 3 min.Vials were microfuged at 13,200 rpm for 5 min and 500 μl of the aqueousphase was then transferred to a fresh tube. These crude samples had somecolored particulate matter in them and some were viscous. These extractswere used in experiments to compare the ability of NESA and PCR todetect bacteria in crude environmental samples. Two sets of purified DNAwere obtained from Lawrence Livermore National Laboratory to be used inthe screening of probes. One set consisted of 96 pools of DNArepresenting 2000 BioWatch filters. The other set consisted of DNAsisolated using the PowerSoil DNA Isolation Kit (MO BIO Laboratories,Inc, Carlsbad, Calif.) from 54 soil samples from geographically diverseregions of the United States.

Whole Genome Amplification of Genomic DNA. Genomic DNAs (10 ng) wereamplified by multiple displacement amplification (MDA) (5) using theREPLI-g Kit from Qiagen (Valencia, Calif.) according to themanufacturer's instructions. Reactions were incubated at 30° C. for 16hours, followed by heat inactivation of the polymerase at 65° C. for 3minutes. MDA DNA was quantified using the PicoGreen assay using themanufacturer's protocol (Invitrogen), and specificity of amplificationby PCR analysis.

Amplification of E. coli genomic DNA in the presence of BioWatchextracts. BioWatch samples were mixed by pipetting up and down to insureresuspension of particulate matter in the samples immediately prior totransfer to a 96-well PCR plate. Approximately, 100,000 E. coli cells,in a suspension of 0.5 μl PBS, were added to each sample. Following theaddition of the cells to the sample, the protocol for “Whole GenomeAmplification from Blood or Cells” (REPLI-g Handbook, Qiagen, January2005) was followed with the following exception: the volume ofnuclease-free water in the master mix was reduced from 27 μl to 24.5 μlto account for the additional volume from the BioWatch samples. MDAreaction products were diluted 40 fold with TE buffer (10 mM Tris-HCl, 1mM EDTA, pH 7.5). One μl of diluted MDA reaction was quantified using aQuant-iT PicoGreen dsDNA Reagent Kit (Invitrogen) according to themanufacturer's instructions. PicoGreen assays were read on a TecanGENios microplate reader. BioWatch samples were tested for the abilityto interfere with the PicoGreen assay by directly adding un-amplifiedBioWatch sample to a PicoGreen assay mix; no interference withquantification was observed.

Locus Representation of BioWatch MDA Samples. MDA reaction products werediluted 4000-fold with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). 1μl of diluted MDA sample was added to a PCR reaction (0.5 units PlatinumTaq polymerase (Invitrogen), 0.3 μM each of forward and reverse primers,0.25 μM TaqMan probe, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 5 mM MgCl2, 1mM dNTPs, 1× Rox reference dye (Invitrogen)). Reactions were performedand quantified with an Applied Biosystems 7300 Real Time PCR systemunder the following conditions: 10 min at 95° C., followed by 40 cyclesof 95° C. for 15 sec and 60° C. for 1 min. The sequences of the PCRprimers and TaqMan probes for the ExoV and OmpA loci have been publishedpreviously (20).

Nicking Endonuclease Signal Amplification (NESA). NESA reactions wereperformed as previously described (11). In brief, 1 pmole fluorescentlylabeled probe, 1 μl NEBuffer 2 (New England BioLabs), 1 to 4 μl MDAgenomic DNA and dH2O to a total volume of 9 μl were added to thin-walledPCR tubes or microplates. Samples were incubated at 95° C. for 10 min todenature the genomic DNA and then equilibrated at 58° C. for 5 min. Oneμl (10 U) of Nt.AlwI (New England BioLabs) was added and the reactionincubated at 58° C. for 1 h. The enzyme was denatured by heating thesamples to 80° C. for 20 min. Reactions were stored at 4° C. untilanalysis by capillary electrophoresis (CE) or at −20° C. for long-termstorage. Multiplex assays for B. anthracis were set up with 1 pmole eachof a chromosomal, pXO1 and pXO2 probe. The reaction was kept at 10 μl byreducing the volume of water added. In the case of large-scalescreening, the NESA reactions were processed using a Beckman Biomek® FXrobotic workstation.

Capillary gel electrophoresis. NESA reactions were analyzed using anApplied Biosystems 3130xl Genetic Analyzer (Foster City, Calif.) using a16 capillary array; electrokinetic injection was used in all cases. Thedistance from the loading point to the detector was 35 cm. POP-6™polymer from Applied Biosystems was used with an injection voltage of1.2 kV and a loading time of 18 seconds. Run voltage was 15 kV for 10minutes; oven temperature was set at 60° C. Prior to loading on the ABI3130xl, the reactions were diluted 100-fold: a 20-fold dilution withwater followed by a 5-fold dilution with formamide. Final loading volumewas 10 μl. A set of 5 Hex™-labeled standards was run with each sample toaid in peak identification, which was performed using GeneMapper®software (Applied Biosystems).

Results. A combination of MDA and NESA reproducibly detects bacterialcontamination in crude (BioWatch) environmental samples. Analysis of theDNA in environmental samples is usually constrained by low levels of DNAand the presence of inhibitors of enzymatic reactions (23). We designedour assay to largely overcome both of these constraints by firstamplifying DNA nonspecifically using MDA (14) and then detectingspecific sequences with NESA (FIG. 31). MDA uses phi29 DNA polymerase toamplify total DNA using random hexamers primers and dNTPs. The productof the reaction is highly concentrated DNA that represents the startingDNA with little loss of complexity (5). This reaction can be performedon crude biological samples such as bacterial cultures and whole bloodwithout the need to first purify DNA (2, 8, 9, 24). It is also highlysensitive, amplifying DNA from as little as one bacterium (13, 20). Aschematic of the MDA reaction is shown in FIG. 1A. To determine whetherMDA was capable of amplifying DNA from crude environmental samples, weprepared extracts from 204 filters obtained from the BioWatch Program bybead beating and centrifugation (Methods). E. coli (100,000 cells) wereadded to either 2 μl or 5 μl aliquots of the extracts and MDA performedin a total volume of 50 μl (the filter extracts represented 4% or 10% ofthe MDA reaction volume respectively). There was no significant effectof adding 4% of the crude extract on the overall synthesis of DNA asmeasured by PicoGreen assays (FIG. 32). However, when the relativevolume was increased to 10%, a significant decrease in overall DNAsynthesis occurred. We then used TaqMan qPCR to markers, OmpA and ExoV.No significant amplification bias of these markers was observed in MDAreactions containing either 4% or 10% levels of the crude samples eventhough the 10% reactions had shown a significant decrease in overall DNAsynthesis. We next tested whether NESA could be performed on the MDAsamples described above that had been amplified in the presence of thecrude environmental samples. MDA DNAs amplified in the presence of 10%of the environmental extracts were subjected to NESA without prior DNApurification (FIG. 3). Of 203 samples tested, one sample essentiallyfailed (0.2% of control), and two samples were inhibited more than 50%(28% and 44%), However, the vast majority of samples showed very littleinhibition and overall the samples with added environmental extractaveraged 125±34% of the control.

qPCR is sensitive to inhibitors in the Environmental Extracts PCRanalysis is known to be particularly sensitive to the presence ofcontaminants. We thus suspected that the crude environmental samples wewere using would act as poor templates for PCR. PCR reactions were setup in which 10% of the PCR reaction consisted of the environmentalextract, however these reactions all failed to give a signal (not shown)and so the experiment was repeated with 4% extract (FIG. 33). Twogenetic markers were used OmpA and ExoV. In both cases, approximately20% of samples (40 OmpA and 42 ExoV) failed to amplify and only 5 to 11%(10 OmpA and 23 ExoV) of samples gave signals above 80% of control. Thisis in contrast to the NESA experiments where greater than 94% (191samples) gave signals over 80% of control.

Development of a multiplex assay for B. anthracis The sensitivity of theMDA/NESA assay and its resistance to environmental contaminants makes itan attractive assay system to detect bacterial contamination. Todemonstrate the utility of the assay, we set out to develop a system fordesigning and screening NESA probes capable of identifying specificmicroorganisms. Our initial goal in this was to develop probes thatcould identify individual strains of B. anthracis.

Probe design—Bioinformatics For the purposes of the bioinformaticsscreen, the Nt.AlwI site was placed 2 bases from the 5′ end of the probe(FIG. 34) placing the restriction site 4 bases 3′ of this. The length ofthe probes varied between 18 and 24 bases. The bioinformatics pipelineis shown in FIG. 34. All Nt.AlwI sites were first identified. For eachsite the DNA sequence was extended 1 base 5′ and 10 bases 3′ and theresulting 16 base sequences were compared to the GenBank (non-redundant)database and identical sequences were discarded. Of the 5286 Nt.AlwIsites in B. anthracis (chromosomal and plasmid sites) 133 were unique.These 133 sequences were extended at the 3′ side to form all thesequences from 18 to 24 bases. Each of these 931 sequences (7 sequencesper unique 16mer) were then compared against the GenBank (non redundant)database and the closest matches ranked using the scoring matrix shownin Table 1. The overall pattern of the scoring matrix was initiallybased on experiments showing the relative importance of sequences in andnear the nicking enzyme recognition site (not shown) but the actualvalues used were arbitrary. These sequences were placed in a searchabledatabase consisting of the probe sequence, % GC content, Tm to B.anthracis target and a list of closest matches in GenBank together withtheir score and Tm (target of 54° C.) and the absence of possibleintermolecular or intramolecular stem-loop structures.

Probe Screening Candidate probes were first tested against three nearneighbors of the B. anthracis genome, B. cereus, B. subtilis, and B.thuringiensis; any candidate probes showing positive results wereeliminated from subsequent screening in order to eliminate falsepositive detection in assays for B. anthracis. In total, all 133 probeswere tested against 14 strains from the 3 near neighbors. Survivingprobes were then tested against the eukaryote and prokaryote MDA panels,including various strains of B. anthracis MDA. Probes showing positiveresults for the B. anthracis strains only and no cross-reactivity withthe background panels were then tested against the 96 MDA pools of 4000environmental aerosol samples, and subsequently the 54 MDA soil samples.All remaining probes were then retested against the B. anthracisstrains, prokaryote and eukaryote panels.

Discrimination between strains of B. anthracis Our large scale screeninganalysis identified a set of probes that could detect B. anthracischromosomal and plasmid sequences but that did not react with any of theDNAs in our screening panels including over 2000 environmental samples.To test whether these probes could be used to genotype unknown samplescorrectly, a blinded experiment was performed. DNA samples from a set ofB. anthracis strains and related Bacillus species were coded, amplified,and subjected to NESA at ECBC. These reactions were then analyzed by CEat Georgetown University Medical School, and the results decoded byECBC. All three related Bacillus species tested gave a positive result(FIG. 35). Furthermore, the plasmid content of the strains wasdetermined accurately in all cases.

Parallel and multiplex NESA For analysis of a large number of probesagainst one or more organisms, a system that is amenable to parallel,and/or multiplex analysis is essential. For instance, in environmentalsampling for pathogens, a whole host of different targets could beenvisioned (1). During our large scale screening, analysis was performedwith 96 reactions in parallel using 96-well plates and a Beckman BiomekFX robotic liquid handling station. The MDA reaction produces a vastexcess of DNA enabling over 100 NESA reactions from a 50-μl MDA reactionand this can be scaled up easily. Although 500 ng of MDA DNA was used inthese screening assays, less than 100 ng of MDA DNA is sufficient forNESA (11). To increase the efficiency of the assay even more wedeveloped a multiplex assay using FAM16 labeled probes specific for B.anthracis chromosomal and plasmid (pXO1 and pXO2) sequences that couldbe distinguished from each other by size using capillaryelectrophoresis. These probes were used in NESA reactions in thepresence of MDA amplified genomic DNA from B. anthracis and relatedBacillus species. Capillary electrophoresis readily distinguishedcleaved and uncleaved probe both for pXO1 and pX02 and chromosomal DNAin the same assay (FIG. 36). For instance, the Ames strain of B.anthracis contains both pX01 and pX02 and thus generates signals fromall three probes (chromosomal, pXO1 and pXO2) (FIG. 36 top). In Δ Ames,which contains only pXO2, the pXO1, signal is absent. Similarly, thesignal for pXO2 is absent in strain NNR-Δ1 that lacks pXO2 (although thepXO1 and chromosomal signals obtained with this strain are lower for anunknown reason). With ΔSterne, a strain that lacks both plasmids, onlythe chromosomal signal was observed.

Discussion We have demonstrated that the combination of MDA and NESA canbe used to develop fast, sensitive and specific multiplex assays fororganism identification. This combination of techniques provides analternative to PCR, which is sensitive to common environmentalinhibitors.

Sensitivity The theoretical sensitivity of our combined MDA/NESA assayis one genome equivalent since it has been shown that MDA can beperformed on one bacterium (13, 20). Detection levels of one bacteriumare likely to be hard to obtain routinely but we have previouslydemonstrated the ability to detect 7 cfu of B. subtilis in crude samples(11).

Assay time The slowest step in the assay is the MDA reaction that isroutinely run overnight as recommended by the manufacturer. Preliminaryresults using the new ultrafast kit from Qiagen indicate that a standardMDA reaction yields sufficient DNA for NESA within 90 min. Data providedby Qiagen indicate that sufficient DNA should be produced within 45 min,but we have yet to confirm this. Further increases in speed can bepossible by supplementing the reaction with additional enzyme(unpublished) or by using semi-specific primers for the region of thegenome under investigation. The sensitivity is also dependent on thetype of target DNA since plasmid DNA is usually amplified better in MDAthan genomic DNA (6). Plasmid DNA can also give a stronger signalcompared to chromosomal DNA due to the higher copy numbers of manyplasmids compared to chromosomal DNA. In the case of B. anthracis,probes against pXO1 and pXO2 usually gave stronger signals thanchromosomal probes.

NESA reactions were run for 1 h here, but have been shown previously tobe nearly complete within 30 min (11). Thus a 30 min NESA should besufficient in all cases except where maximum sensitivity is required.Following NESA, CE separation is complete within 20 min. The use of a16-capillary machine and a triplex assay would put the through rate at48 probes in 20 min.

Multiplex Capability We have demonstrated that it is possible tomultiplex NESA reactions using size discrimination to detect B.anthracis chromosomal, and plasmid (pXO1 and pXO2) DNA in the sameassay. Multiplex assays can be designed using different fluors (20), orby placing the fluor on either the 5′ (as in this paper) or the 3′ end(unpublished) of the probe. Although discrimination by size on CE oftenhas a resolution of less than one base, the small size of thefluorescent oligos used in NESA results in migration that depends onbase composition as well as overall length. Multiplex assays must beoptimized with regard to the migration pattern of the cleaved probessince fluorescent oligos of different sizes can run at the sameposition, oligos of the same length can separate into two distinct peaksand oligos of the same length and base composition can resolve intoseparate peaks when labeled with different fluors (20). For instance,the pXO1 and chromosomal probes are cleaved into fluorescentoligonucleotides of identical length yet they resolve on CE (FIG. 6).

Bioinformatics of Probe Selection We designed our bioinformaticspipeline to first identify short regions (24mers) that contained Nt.AlwIsites in B. anthracis and then selected against those regions thatcontained an identical 16 base region, anchored by the Nt.AlwI site, inGenBank. This screening procedure was computationally intense andresulted in a relatively small number of potentially acceptable targets(approximately 2.5%). However, remarkably 17% of these regions yieldedprobes with absolute specificity in the screening assays conductedsuggesting that probe development against other targets is feasible.Indeed, preliminary results with Y. pestis chromosomal probes indicate asimilar success rate. It should be noted that each Nt.AlwI site canyield multiple probes so that the 136 unique probes analyzed are only asmall fraction of what is available. In recent unpublished work, we havesuccessfully made probes against reverse transcribed MS2 bacteriophage(3.85 kb) and Dengue virus genomic RNA (˜10.7 Kb) indicating that theMDA/NESA technique can be used on small genomes.

FIG. 31 Design of the assay The overall design of this exemplary assayis shown in A. DNA first undergoes nonspecific whole genomeamplification using MDA (5) (B). Following a 95° C. denaturation stepthe isothermal MDA reaction yields highly concentrated single and doublestranded amplified DNA of high molecular weight (Dean et al. 2001). Asmall fraction of this amplified DNA is then used for specific analysisusing NESA (C). MDA DNA is denatured at 95° C. followed by a 58° C.isothermal reaction in which a probe containing a single strandedendonuclease (Nt.AlwI) site anneals to single stranded target DNA and iscleaved by the restriction enzyme. The target remains uncleaved so thatupon dissociation of the cut probe, fresh, full-length probe can annealand be cleaved by the enzyme. This repeated annealing and cleavage ofthe probe generates a linear amplification of signal from each targetsequence (figure adapted from (11).). Cleaved probe is quantified usingCE.

FIG. 32 MDA can amplify DNA from environmental collection filters. E.coli (105 cells) in 0.5 μl were added to either 2 μl or 5 μl of 204BioWatch samples (4% and 10% respectively of the final reaction volumeof 50 μl), which were then subjected to MDA. The yield of DNA from theMDA reaction was quantified by PicoGreen assay and compared to controlswith 5 μl TE instead of the environmental sample (n=29). Locusrepresentation was determined by TaqMan qPCR on 2 loci, ExoV and OmpAand compared to TE controls (n=29). The upper and lower limits of thebox represent the 75th and 25th percentiles, the line within the box isthe median value, the filled square is the mean, and the whiskersrepresent the 90th and 10th percentiles. *Significantly lower than thecontrol p<0.001 (t-test).

FIG. 33 Efficient detection of genomic DNA in crude environmentalsamples by NESA but not by qPCR. The MDA samples generated in FIG. 2were subjected to NESA (left two panels). The standard 10 μl NESAcontained either 1 μl BioWatch sample or 1 μl TE. qPCR reactions wereset up containing 4% BioWatch sample as in FIG. 2 and analyzed by qPCRfor the OmpA and ExoV markers. The histogram compare the frequency as apercentage of the total with the ranges shown on the figure: 0represents the number of reactions that gave no detectable signal, >represents reactions that have values above 180, the other valuesrepresent the upper limit of a 20% range. Black bar, no signal; greybar, <80% control; white bar, >80% control.

FIG. 34 Probe screening. A. Probe structure and bioinformatics screen.Probes contained an Nt.AlwI site 2 bases from the 5′ end of the probe.When the probe binds to complementary DNA, Nt.AlwI cuts after 4 bases 3′of the recognition site. The overall length of the probes varied between18 and 24 bases. The bioinformatics screen yielded a database of 931potential probes that had a unique 16-base core region. The probes wereordered using a scoring matrix (Table 1) based, in part, on mismatchmutational analysis (not shown). B. Probe validation (experimentalscreening). Potential probes were screened against MDA genomic DNA asshown in the figure and in Methods. The final 23 probes recognize B.anthracis DNA and have never cross-reacted with any other genomic DNA

FIG. 35 Characterization of probe activity on B. anthracis strains andon related species of Bacillus Genomic DNA from the Ames strain of B.anthracis (our positive control) was amplified by MDA and then analyzedby NESA. Nine genomic DNAs representing 6 B. anthracis strains and 3related Bacillus species underwent MDA and NESA at Edgewood Chemical andBiological Command as detailed in Methods except that 4 μl of the MDAreaction was used for NESA. These samples were encoded and then analyzedby CE at Georgetown University. The samples were scored for the presenceof B. anthracis chromosomal, pXO1 and pXO2 DNA using probes specific forthese 3 genetic elements and then the samples were decoded. Thegenotypes of the B. anthracis strains used are shown.

FIG. 36 Performance of a multiplex assay for B. anthracis detection andgenotyping Probes for B. anthracis chromosomal, pXO1 and pXO2 DNA wereused simultaneously in NESA reactions on MDA DNA from the B. anthracisstrains indicated (methods). The 5-base and 17-base size markers areshown flanking the trace. The uncut probes for chromosomal, pXO1 andpXO2 eluted at 24, 23 and 21 bases The signals from chromosomal, pXO1and pX02 DNA eluted at 24, 23 and 20 bases (not shown) and the cutprobes at 10, 9 and 11 bases respectively. The cut pXO1 probe eluted ataround 9 bases even though its actual size is 10 bases. The 5mer markerwas used as an internal control; the other signals were scaledaccordingly.

TABLE 4 Scoring Matrix Position Score 1 Not scored 2 0.3 3-7 Not scored8 1   9 0.8 10 0.9 11 0.7 12 0.3 13 0.3 Other positions 0.1

The probe design is shown in FIG. 34. Positions 3-7 are the Nt.AlwIsite, they are not scored because they have to have an exact match (onlyexact matches in this region make it through to the scoring matrix).

References for Example 8

-   1. 2007, posting date. U.S. Government Lists of Bioterrorism Agents    and Diseases. Federation of American Scientists. [Online.]-   2. Abulencia, C. B., D. L. Wyborski, J. A. Garcia, M. Podar, W.    Chen, S. H. Chang, H. W. Chang, D. Watson, E. L. Brodie, T. C.    Hazen, and M. Keller. 2006. Environmental whole-genome amplification    to access microbial populations in contaminated sediments. Appl    Environ Microbiol 72:3291-301.-   3. Belec, L., J. Authier, M. C. Eliezer-Vanerot, C. Piedouillet,    1 A. S. Mohamed, and R. K. Gherardi. 1998. Myoglobin as a polymerase    chain reaction (PCR) inhibitor: a limitation for PCR from skeletal    muscle tissue avoided by the use of Thermus thermophilus polymerase.    Muscle Nerve 21:1064-7.-   4. Bickley, J., J. K. Short, D. G. McDowell, and H. C. Parkes. 1996.    Polymerase chain reaction (PCR) detection of Listeria monocytogenes    in diluted milk and reversal of PCR inhibition caused by calcium    ions. Lett Appl Microbiol 22:153-8.-   5. Dean, F. B., S. Hosono, L. Fang, X. Wu, A. F. Faruqi, P.    Bray-Ward, Z. Sun, Q. Zong, Y. Du, J. Du, M. Driscoll, W.    Song, S. F. Kingsmore, M. Egholm, and R. S. Lasken. 2002.    Comprehensive human genome amplification using multiple displacement    amplification. Proc Natl Acad Sci USA 99:5261-6.-   6. Dean, F. B., J. R. Nelson, T. L. Giesler, and R. S. Lasken. 2001.    Rapid amplification of plasmid and phage DNA using Phi 29 DNA    polymerase and multiply-primed rolling circle amplification. Genome    Res 11:1095-9.-   7. Galluzzi, L., M. Magnani, N. Saunders, C. Harms, and I. J.    Bruce. 2007. Current molecular techniques for the detection of    microbial pathogens. Sci Prog 90:29-50.-   8. Gonzalez, J. M., M. C. Portillo, and C. Saiz-Jimenez. 2005.    Multiple displacement amplification as a pre-polymerase chain    reaction (pre-PCR) to process difficult to amplify samples and low    copy number sequences from natural environments. Environ Microbiol    7:1024-8.-   9. Hosono, S., A. F. Faruqi, F. B. Dean, Y. Du, Z. Sun, X. Wu, J.    Du, S. F. Kingsmore, M. Egholm, and R. S. Lasken. 2003. Unbiased    whole-genome amplification directly from clinical samples. Genome    Res 13:954-64.-   10. Jannes, G., and D. De Vos. 2006. A review of current and future    molecular diagnostic tests for use in the microbiology laboratory.    Methods Mol Biol 345:1-21.-   11. Kiesling, T., K. Cox, E. A. Davidson, K. Dretchen, G. Grater, S.    Hibbard, R. S. Lasken, J. Leshin, E. Skowronski, and D. Danielsen.    Submitted. Sequence specific detection of DNA using nicking    endonuclease signal amplification (NESA). Nucleic acids research.-   12. LaMontagne, M. G., F. C. Michel, Jr., P. A. Holden, and C. A.    Reddy. 2002. Evaluation of extraction and purification methods for    obtaining PCR-amplifiable DNA from compost for microbial community    analysis. J Microbiol Methods 49:255-64.-   13. Lasken, R. 2005. Multiple displacement amplification from single    bacterial cells, p. 119-147. In S. L. R. Hughes (ed.),    Amplification: Methods Express. Scion Publishing Ltd, Oxford.-   14. Lasken, R. 2005. Multiple displacement amplification of genomic    DNA, p. 99-118. In S. L. R. Hughes (ed.), Amplification: Methods    Express. Scion Publishing Ltd, Oxford.-   15. Makino, S., and H. I. Cheun. 2003. Application of the real-time    PCR for the detection of airborne microbial pathogens in reference    to the anthrax spores. J Microbiol Methods 53:141-7.-   16. Monis, P. T., and S. Giglio. 2006. Nucleic acid    amplification-based techniques for pathogen detection and    identification. Infect Genet Evol 6:2-12.-   17. Mothershed, E. A., and A. M. Whitney. 2006. Nucleic acid-based    methods for the detection of bacterial pathogens: present and future    considerations for the clinical laboratory. Clin Chim Acta    363:206-20.-   18. Mumy, K. L., and R. H. Findlay. 2004. Convenient determination    of DNA extraction efficiency using an external DNA recovery standard    and quantitative-competitive PCR. J Microbiol Methods 57:259-68.-   19. Peters, R. P., M. A. van Agtmael, S. A. Danner, P. H. Savelkoul,    and C. M. Vandenbroucke-Grauls. 2004. New developments in the    diagnosis of bloodstream infections. Lancet Infect Dis 4:751-60.-   20. Raghunathan, A., H. R. Ferguson, Jr., C. J. Bornarth, W.    Song, M. Driscoll, and R. S. Lasken. 2005. Genomic DNA amplification    from a single bacterium. Appl Environ Microbiol 71:3342-7.-   21. Roh, C., F. Villatte, B. G. Kim, and R. D. Schmid. 2006.    Comparative study of methods for extraction and purification of    environmental DNA from soil and sludge samples. Appl Biochem    Biotechnol 134:97-112.-   22. Sachse, K. 2004. Specificity and performance of PCR detection    assays for microbial pathogens. Mol Biotechnol 26:61-80.-   23. v. Wintzingerode, F., U. B. Gobel, and E. Stackebrandt. 1997.    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Example 9 Application of Nicking Endonuclease Signal Amplification(NESA) to the Detection of RNA Genomes: Development of a Multiplex Assayfor all Four Serotypes of Dengue Virus

(References cited in this example are listed at the end of thisexample.)

Sequence-specific genomic assays for the detection and typing ofmicroorganisms using procedures such as PCR are common. However, in thecase of RNA genomes, their small size and the requirement to first copythe RNA into cDNA often limits their sensitivity. We have developed anew assay for organisms with RNA genomes that uses a combination ofreverse transcription, multiple displacement amplification (MDA) andnicking endonuclease signal amplification (NESA). This assay, which weterm rNESA, achieves high sensitivity, differentiates between viralserotypes, and is insensitive to common environmental and biologicalcontaminants. Using the small RNA virus surrogate MS2 bacteriophage, wewere routinely able to detect 100 attograms of genomic RNA with rNESA.At 10 attograms (5 genomes) the NESA assay detected MS2 approximately40% of the time. With the same samples, a published rtPCR assay had adetection threshold of 10 fg genomic RNA (5,000 genomes). The presenceof a large excess of either human mRNA, or environmental contaminants,had no effect on the specificity of the assay and only a small effect onsensitivity. The utility of the assay for larger RNA viruses was shownby the development of a rNESA multiplex assay specific for the fourserotypes of the human pathogenic Dengue virus. These probes hadabsolute specificity between the Dengue virus serotypes and no crossreactivity with human RNA or with two other members of the Flaviviridaefamily, West Nile virus and St Louis Encephalitis virus. The Dengueassay detected 2 fg Dengue virus RNA (˜345 genome equivalents).

Detection and identification of microorganisms is critical in the areasof medical diagnostics, biodefense and environmental biology. Manydetection methods have been used including selective culture in vitro,animal inoculation, mass spectrometry, antibody-dependant assays andassays that use DNA sequence-specific hybridization (Ivnitski et al.2003; Lim et al. 2005; Peruski and Peruski 2003; Rotz and Hughes 2004).Of the DNA-based techniques, PCR is perhaps most often used because ofits high specificity, speed, and its adaptability to new targets. RNAviruses, however, are an exception because there is an additionalrequirement to first convert the RNA to DNA. This first step, which usesreverse transcriptase, is inefficient and limits the sensitivity of theoverall assay (Bustin and Nolan 2004). In qPCR, a relatively smalltarget DNA sequence is amplified using a gene-specific primer pair insequential rounds of denaturation, annealing, and DNA synthesis using aheat tolerant DNA polymerase. The product of the reaction is detectedusing fluorescent probes. That is, amplification and detectionessentially occur at the same time. We recently separated these twosteps by amplifying DNA nonspecifically using MDA and then detecting thepresence of specific sequences using NESA (Kiesling et al. 2007). InNESA, a single stranded fluorescent probe containing a nickingendonuclease recognition sequence anneals to the target sequence forminga double stranded recognition and cleavage site. The nickingendonuclease then cleaves the probe but leaves the target intact. Thesmall probe pieces dissociate from the target spontaneously allowingmultiple rounds of hybridization and cleavage to occur. The cleavedprobe can be detected in a number of ways although we usually usecapillary electrophoresis. We have also used FRET-based probes wherefluor and quencher are separated upon DNA cleavage (unpublished). Arecent paper used cleavage of molecular beacons in their NESA reaction(Li et al. 2008). In this paper, we describe an MDA-NESA protocol(rNESA) that allows detection of RNA viral genomes with a sensitivitythat outperforms rtPCR. Remarkably, the assay can also be performeddirectly on environmental samples such as extracts of BioWatch filtersthat are refractory to PCR analysis. BioWatch is a US government programthat monitors air for biological threat agents using filtration (Report2003). We demonstrate the power of rNESA by developing a multiplex assayagainst the four Dengue virus serotypes. Probes against each of the fourDengue serotypes were specific for the respective serotype and did notcross react with other closely related RNA viruses. In addition, theDengue virus rNESA assays were able to detect low levels of Dengue RNAeven in the presence of a large excess of nonspecific (human) RNA.

Results

Detection of genomic RNA using reverse transcriptase and NESA For theinitial experiments, we used MS2 bacteriophage genomic RNA as a target.MS2 is often used as an RNA virus surrogate since there is no risk ofinfection and it can be handled at biosafety level I (O'Connell et al.2006). MS2 first strand cDNA was reverse transcribed as detailed inmethods. This cDNA was then used in a series of NESA reactions usingNESA probes designed to hybridize to either the cDNA (MS2-S) or to theRNA strand itself (MS2-1). As expected, the MS2-S probe recognized thetarget cDNA and was cleaved by Nt.AlwI as shown by the production ofcleaved probe (FIG. 1A). Under these conditions at least 0.1 ng ofstarting RNA were required. In contrast, the antisense probe MS2-1failed to detect MS2 RNA either before or after cDNA synthesis. Theseresults were expected since Nt.AlwI is not known to cleave RNA-DNAhybrids. In an attempt to increase the sensitivity of the assay, MS2cDNA was nonspecifically amplified by MDA and then subjected to NESA.MDA was felt to be a near ideal amplification method since it had beenshown previously that MDA using random primers results in lowamplification bias and very little loss of specific sequences (Hosono etal. 2003). MDA has even been used to genotype a single bacterium(Raghunathan et al. 2005). The addition of MDA did indeed result inincreased NESA sensitivity such that 100 ag of MS2 could be reliablydetected (FIG. 1B). At 10 ag, detection varied between replicates. Forinstance, in FIG. 1B one of three replicates gave a detectable signal.In all, we have performed the analysis on 10 ag MS2 RNA 17 times; asignal was observed in 7 of these, that is approximately 40% of thetime. 10 ag corresponds to approximately 5 MS2 genomes. This level ofsensitivity is remarkable for an assay using reverse transcriptase. Inorder to directly compare rNESA with rtPCR, the same MS2 dilutions usedin FIG. 1B were subjected to rtPCR with two different primer sets usinga recently published protocol (O'Connell et al. 2006). rtPCR reliablydetected 10 fg MS2 RNA in this experiment (FIG. 1C). Melting curveanalysis indicated specific amplification at 10 fg but not 1 fg ofstarting RNA. This value is in agreement with the limits of detectionshown previously using these primers and it agrees with other primersets that have been reported independently (Dreier et al. 2005; Rolfe etal. 2007).

rNESA is resistant to inhibitors found in crude environmental samplesDetection of genomic DNA or RNA in environmental samples usuallyrequires purification of the genomic material in order to removecontaminants that inhibit enzymatic reactions. In the case of PCR,inhibitors can cause loss of specific signal resulting in falsenegatives and/or the inappropriate amplification of non-target DNAresulting in false positives (Bustin and Nolan 2004; Lantz et al. 2000;Radstrom et al. 2004). Both scenarios are unacceptable in programs, suchas BioWatch (Report 2003), that are designed to detect the release ofbiowarfare agents in American cities. To compare the effects ofcontaminants found in crude BioWatch samples on rtPCR and rNESA, aseries of MS2 rtPCR and rNESA reactions was set up containing 100 fg MS2genomic RNA and 10% (v/v) of BioWatch sample. All 14 independentBioWatch extracts inhibited the amplification of MS2 genomic RNA byrtPCR and six of these extracts caused the rtPCR assay to failcompletely (FIG. 2A). The BioWatch samples also inhibited rNESA to someextent, but this inhibition was relatively minor (24%) and the rNESAsignal was not lost with any BioWatch sample (FIG. 2B). That is, theBioWatch extracts caused dropout (false negatives) in the rtPCRreactions but not in the rNESA reactions.

Sensitivity in a complex background (selectivity) One concern is that alarge excess of non-target RNA could decrease rNESA's ability toefficiently amplify and detect some minute quantity of target RNA. Totest selectivity, decreasing levels of MS2 genomic RNA (100 fg to 100ag) were added to rNESA reactions containing 10 or 100 pg of purifiedtotal human RNA (FIG. 2C). The MS2-1 probe did not give a signal withhuman RNA alone as expected. 10 pg of human RNA had little effect onrNESA's ability to detect even the smallest concentration of MS2 RNA(100 ag). Thus rNESA can detect MS2 in the presence of a 100,000-foldexcess (w/w) of non-specific RNA. In the presence of 100 pg of humanRNA, NESA could again detect MS2 RNA in the presence of a 100,000-foldexcess of human RNA but the absolute limit of detection changed to 1 fg(FIG. 2C).

rNESA probes specific for Dengue virus serotypes To demonstrate thatrNESA can be used to detect pathogenic RNA viruses we set out to developprobes specific for each of the 4 Dengue virus serotypes (Chambers etal. 1990). The sequence of the 4 serotype probes and their relationshipto the 4 viral serotypes is shown in (FIG. 3A). Each serotype-specificprobe was used in a series of rNESA reactions containing 100 fg genomicRNA from each of the viral serotypes. All four serotype-specific probes1, 2, 3 and 4 were completely specific for DEN-1, DEN-2 and DEN-3, andDEN-4 respectively with no cross reactivity with other serotypes. Thespecificity of these four probes is further shown by the fact that theydo not react with two other members of the Flavivirus genus, West Nilevirus and Saint. Louis Encephalitis virus. Since the Dengue probes weredeveloped to detect and type Dengue virus in human RNA samples, werepeated the Dengue serotype rNESA assays in the presence of 10 pg or100 pg of total human RNA (FIG. 4 b). As expected, none of the fourprobes interacted with the human RNA. In addition, added human RNA hadlittle effect on the ability of the probes to detect 100 fg Dengue RNA.

Development of a multiplex Dengue virus assay Initial experiments withthe four serotype-specific probes showed that although the signal peaksresolve by capillary electrophoresis (CE), they are not well separatedand had some overlap. To obtain signals that are fully separated on CE,two new probes (3T and 4T) were generated that had additional non basepairing residues (T's) at their 3′ ends (Table 1) and thus yield largerprobe fragments (FIG. 5). All four probes were used in a multiplex rNESAreaction and the signals resolved by CE. All four peaks were distinctfrom each other. In order to identify peaks and ensure that there was nocross reactivity, a series of reactions were performed that lacked justone of the serotypes. All of the probes could be mapped to a specificpeak and there was no cross reactivity (FIG. 5). The sensitivity of theDengue assay was compared to a published qPCR assay (FIG. 6). Our DenguerNESA assay detected 2 fg Dengue RNA (approximately 345 Dengue virusgenome equivalents) whereas the qPCR assay on the same Dengue virusdilutions had a lower limit of detection of 20 fg. Melting curveanalysis showed that there was no detectable specific amplification inthe control and 2 fg PCR experiments.

Discussion We have described a new technique that can be used to developspecific assays for the detection of organisms with RNA genomes. Thetechnique, which is a combination of reverse transcription, MDA andNESA, retains the properties of the individual techniques. For instancethe inhibitor-resistant phi29 DNA polymerase in MDA allows detection indirty backgrounds and fast, nonspecific whole genome amplification whilethe moderate temperature resistance of Nt.AlwI (up to 60° C.) allows thedevelopment of specific DNA probes. Remarkably, the assay is moresensitive than rtPCR both for the small (3.579 kb) virus substitute, MS2and for the larger (˜10.7 kb) Dengue virus.

Sensitivity We have shown that rNESA can detect 100 ag of MS2 RNAroutinely and 10 ag about 40% of the time. 10 ag is equivalent toapproximately 5 genomes. A surprising finding was that rNESA was moresensitive than published rtPCR MS2 assays (Dreier et al. 2005; O'Connellet al. 2006; Rolfe et al. 2007). This is probably due to the nature ofthe cDNA synthesized in the reverse transcriptase step. Reversetranscription is dependent on an initial DNA primer that initiates cDNAsynthesis. Since the ability of primers to initiate productive cDNAsynthesis is highly dependent on the location of hybridization due tofolding of the RNA, random primers are often used to sample manyinitiation sites (Bustin and Nolan 2004). Although this results inrelatively efficient cDNA synthesis, most cDNA molecules are not fulllength. This reduces the ability of PCR to amplify the cDNA because somecDNA molecules will not contain sequences having both forward andreverse priming sites. These sequences will not act as templates for PCRand thus limits of detection will decrease. This is not the case forrNESA because neither amplification of the cDNA with MDA, noramplification of the signal with NESA, requires a primer pair. It ispossible that the NESA target sequence (approximately 20 bases) can besplit in two resulting in loss of signal but this will occur far lessfrequently than separation of the forward and reverse primers of rtPCRthat can be 100 or more nucleotides apart. For the same reason, NESA canbe less sensitive to degradation of the RNA during analysis. The Denguevirus assay was not as sensitive as the MS2 assay. We do not know ifthis is related to the larger size of the Dengue virus. However, theDengue rNESA assay was still approximately 10-fold more sensitive thanthe qPCR assay.

Specificity We have previously shown that NESA probes can be used todistinguish between bacterial species (Kiesling et al. 2007). Here, weshow that it is possible to develop specific probes against the closelyrelated serotypes of Dengue virus. These probes not only distinguishbetween Dengue serotypes but they also do not interact with West Nilevirus and St. Louis Encephalitis virus that are also members of theFlavivirus genus. Both the Dengue and MS2 probes are also refractive tototal human RNA. We are currently developing probes that are specificfor groups of organisms. For instance we are developing a probe thatrecognizes all Dengue virus serotypes and another that recognizes manyof the members of the Flavivirus genus.

Selectivity We show that rNESA selectively detects 100 ag of MS2 genomicRNA (˜50 genomes) in the presence of 10 pg total human RNA. 10 pg ofhuman RNA is the approximate amount of RNA in 2 to 10 cells, thus theassay can theoretically detect the equivalent of 5 viral genomes perhuman cell. With 100 pg of human RNA there was a decrease in sensitivityof the assay presumably due to competition between the human RNA and MS2RNA for reverse transcriptase. It would be interesting to see if the useof MS2-specific reverse transcriptase primers results in increasedsensitivity in the presence of such a large excess of human RNA.

Application to environmental monitoring and medical diagnostics In bothof these applications, samples are often contaminated with potentialinhibitors. Heme in blood samples, for instance, is a well-knowninhibitor of PCR as is humic acid from soil in environmental assays(Radstrom et al. 2004). For this reason, samples often undergo extensivepurification before enzymatic analysis. We have shown that crudeenvironmental samples that are refractory to rtPCR analysis can beassayed with rNESA with little loss of sensitivity and no drop out. Thatis, rNESA is particularly resistant to enzymatic inhibitors and thus thegenomic RNA does not need to be extensively purified.

Dengue Multiplex Assay The Dengue multiplex assay we have describeddiscriminates between all four dengue serotypes, does not interact withthe related viruses SLE and WNV, and is applicable to samples containinghuman RNA. The assay should thus be useful to detect acute Dengue virusinfection since detectable viral RNA coincides with the onset ofviraemia. Saxena In our rNESA Dengue multiplex assay we can detect 345genome equivalents. Saxena et al. (Saxena et al. 2008) recentlydescribed a multiplex assay for Dengue serotypes with a sensitivity of2,500 copies. However, it is hard to directly compare the two assayssince the assay described by Saxena et al (Saxena et al. 2008) is basedupon in vitro transcription of a small region of the virus compared toour full-length assays, and supporting evidence for their sensitivitydata is not shown. The apparent increased sensitivity of the rNESAmultiplex assay over this rtPCR multiplex assay warrants furtherinvestigation using clinical samples. Indeed, the ability to use crudeRNA samples, and the easily automated MDA and NESA reactions(unpublished) would make this an attractive diagnostic assay.

Non-viral RNA targets Although the work presented in this paper targetsviral RNA, the technique we describe should be amenable to any RNAtarget that can be reverse transcribed. Indeed, the sensitivity of theassay is such that detection of mammalian mRNA targets should bepossible.

Methods

Oligonucleotides, genomic RNA and Nt.Alw1 Oligonucleotides (Table 1)were from Integrated DNA Technologies (Coralville, Iowa,) except theβ-actin PCR primers (Stratagene). Probes contained a FAM group at eitherthe 5′ or 3′ end and were purified by RNAse-free ion exchange HPLC.Oligonucleotide size standards were synthesized withhexachlorofluorescein (HEX™) at their 5′ ends. MS2 genomic RNA wasobtained from Roche Applied Science (Indianapolis, Ind.). Dengue virusserotype-3, West Nile virus, and St Louis Encephalitis virus genomicRNAs were obtained from BEI resources (Manassas, Va.). Human total RNAwas from Stratagene (Cedar Creek, Tex.). Dengue viral Serotypes 1, 2,and 4 genomic RNAs were synthesized by in vitro transcription usingpRS424 plasmids containing full-length genomic cDNA inserts (Polo et al.1997; Puri et al. 2000). Briefly, the plasmids were linearizedimmediately after the last stop codon using SacI. Linearized plasmidswere gel purified using Qiagen Qiaex II Gel Extraction Kit. 2 pg ofpurified linear plasmid was used with the Ambion SP6 Megascript Kit. Thein vitro transcription reactions were incubated for 4 hrs following themanufacturers instructions. The in vitro transcribed RNA was purifiedusing Qiagen RNeasy Mini Kit. Purified RNA was run on a 0.7% agarose gelfor determination of correct size and lack of degradation. RNAconcentrations were determined by A260 and with the Invitrogen MolecularProbes Quant-it RiboGreen RNA Assay Kit. Serotypes of the four Denguevirus RNA samples were confirmed using serotype specific primers inrtPCR (Saxena et al. 2008).

Reverse Transcription RNA was reverse transcribed using the InvitrogenSuperScript III first strand synthesis kit following manufacturersprotocol with slight adjustments. Briefly 2 μl of the RNA was added tothe 8 μl annealing reaction containing 15 μM random hexamers (NewEngland BioLabs), and 1 μl of the annealing buffer, incubated at 65° C.for 5 min, and placed on ice for 2 min. 2 μl of the reversetranscriptase and 10 μl of the 2× reaction buffer were added to the 8 μlannealing reaction while on ice. The 20-μl reaction was heated to 25° C.for 10 min, 45° C. for 50 min, followed by 95° C. for 5 min. 10 μl ofthe 20-μl reaction were used in the proceeding ligation reactions.

Ligation and MDA Ligation and MDA were performed using the Qiagen WholeTranscriptome Kit following the manufacturers instructions except thatan additional enzyme inactivation step (95° C. for 5 min) was addedfollowing the completion of the ligation reaction. The MDA reaction wasperformed for eight hours

NESA Reaction 2 μl of the 50-μl MDA reaction were used in each 10-μlNESA reaction. The NESA reaction contained 1 μl Restriction EndonucleaseBuffer-2 (NEB), 1 μl (1 pmol) probe, 2 μl of target MDA, 5 μl H2O. The9-μl mixture was heated to 95° C. for 10 min, 4° C. for 10 sec, then 58°C. for 5 min. Once the reaction had reached 58° C., 1 μl Nt.AlwI (NEB,Ipswich, Mass.) was added followed by incubation at 58° C. for 60 minand then 80° C. for 20 min to stop the reaction.

Capillary gel electrophoresis Samples were analyzed using an AppliedBiosystems 3130xl Genetic Analyzer with electrokinetic injection asdescribed previously (Kiesling et al. 2007). In brief, NESA reactionswere diluted 100-fold with formamide and then 10 μl were used forinjection. A set of Hex™-labeled standards (5, 17, 21, 30, and 50mer)was run with each sample to aid in peak identification.

rtPCR and Melting Curve Analysis 2 μl of RNA was mixed with 20 μM ofeach forward and reverse primer, 25 μl 2× BioRad iScript One-Step SybrGreen Mix, 2 μl of Reverse Transcriptase, and RNAse-free H2O for a totalreaction volume of 50 μl. Thermocycling parameters were as follows forthe following RNA templates: MS2 RNA: 48° C. for 10 min, 95° C. for 5min, 40 cycles of 95° C. for 10 sec, 62° C. for 30 sec 72° C. for 30sec, Dengue viral RNA: 50° C. for 10 min 95° C. for 5 min 40 cycles 95°C. for 10 sec 55° C. for 30 sec 72° C. for 30 sec, Human RNA: 94° C. for5 min 60° C. for 5 min 72° C. for 1.5 min 35 cycles of 94° C. for 45 sec60° C. for 45 sec 72° C. for 1.5 min followed by final extension of 72°C. for 10 min. All completed rtPCR runs were followed by a melting curveanalysis consisting of the following steps: 95° C. for 1 min 55° C. for1 min, followed by 80 cycles starting at 55° C. for 10 sec increasing in0.5° C. increments each cycle. All rtPCR experiments were run andanalyzed using the BioRad iQ5 RT-PCR Detection System with iQ5 OpticalSystem Software v1.1.

FIG. 37 rNESA MS2 assay is more sensitive than rtPCR. A. MS2 RNA wasreverse transcribed and subjected to NESA using either a probe specificfor the cDNA (MS2-S) or for RNA itself (MS2-1). The sample marked RNAalone was not reverse transcribed. B. MS2 RNA was reverse transcribedand amplified by MDA. 2 μl of the MDA reaction were used in NESA withthe MS2-1 probe. C. The MS2 RNA dilutions used in A were amplified usingtwo sets of published primers (Table 1) (O'Connell et al. 2006). N/A, nospecific amplification was detected. D. The rtPCR reactions in C weresubjected to melting curve analysis. Reactions with starting RNA levelsof 10 fg of RNA or more had similar profiles and only the 10 fg reactionis shown. Neither the no RNA control nor the 1 fg reaction haddetectable specific amplification. All reactions were performed intriplicate; the standard deviation is shown. *Significantly differentfrom the no RNA control (T-Test, p<0.01)

FIG. 38. rNESA but not rtPCR is resistant to environmental contaminants.A. rtPCR reactions were set up using 100 fg MS2 RNA and either primerset S2 or S3.10% of the final volume of the reaction contained either TE(−) or an independent crude BioWatch extract (1 to 14). B. rNESAreactions were set up using 100 fg MS2 RNA and probe MS2-1.10% of thefinal volume of the reaction contained either TE (−) or an independentcrude BioWatch extract (1 to 14). C. rNESA reactions were set upcontaining decreasing concentrations of MS2 RNA and the MS-1 probe andeither 1 or 10 pg of human RNA. All reactions were performed intriplicate; standard deviation is shown. *Significantly different(T-Test) from the no RNA control (p<0.01).

FIG. 39. Dengue virus genome organization and probe location. Locationsof selected nicking endonuclease recognition sites are shown relative tothe gene structure of the Dengue genome. The two regions used forNt.AlwI probe development are shown together with the RNA sequence ofthe four Dengue serotypes. Underlined blue and green underlined regionswere used as sense and antisense DNA probes respectively (Table 1).Nt.AlwI sites are shown in red. AnchC, anchored capsid protein; prM,membrane precursor protein; E envelope (E) protein; NS, nonstructuralprotein (1, 2A, 2B, 3, 4A, 4b, 5).

FIG. 40. Dengue virus serotype-specific rNESA assay. A. Eachserotype-specific probe (probes 1 to 4) were used independently in a setof rNESA reactions containing no RNA (−), 100 fg genomic RNA from Dengueserotypes 1 to 4 (1−4), St Louis Encephalitis virus (SLE) or West Nilevirus (WNV) genomic RNA. B. Each serotype-specific Dengue probe was usedwith its cognate Dengue RNA in rNESA reactions containing 0, 10 or 100pg total human RNA.

FIG. 41. Dengue multiplex assay Dengue serotype-specific multiplexprobes (Dengue 1, 2, 3T, 4T) were used in a multiplex assay containingall four Dengue serotypes (first panel) or in assays missing one of theserotypes. The peak locations corresponding to each probe are shownunderneath traces of the capillary electrophoresis analysis. The datahas been normalized to a 5 by HEX-labeled size and loading controloligonucleotide (not shown).

FIG. 42. Sensitivity of the Dengue rNESA assay Serial dilutions ofDengue serotype 4 RNA were subjected to rNESA using the DEN4 probe (A)or to rtPCR using primers described by Saxena et al.(Saxena et al. 2008)(B). All reactions were performed in triplicate; averages and standarddeviations are shown. After rtPCR, samples were subjected to meltingcurve analysis; the absence of specific amplification is denoted by N/A.*, significantly different from control (T-Test, p<0.01).

TABLE 5 Oligonucleotide Sequences NESA Probes MS2-15′-FAM-TGGATCTGACATACCTCCGA-3′ MS2-S 5′-CCGTGGATCAGACACGC-FAM-3′Dengue-1 5′-CGTACTAGGATCACAAGAAGGA-FAM-3′ Dengue-25′-FAM-TTGGATCATAGGGTATTGGATCTA-3′ Dengue-35′-GTTGTCCTTGGATCGCAAGAGGGA-FAM-3′ Dengue-3T5′-GTTGTCCTTGGATCGCAAGAGGGATT-FAM- 3′ Dengue-45′-AGTGCTGGGATCTCAGGAAGGA-FAM-3′ Dengue-4T5′-AGTGCTGGGATCTCAGGAAGGATTTT-FAM- 3′ PCR Primers MS2 Set 2 F:5′-TGCGTTGCGTAAAGGCGATGAAGA-3′ R: 5′-TTTCCGGCACCAGGTTAATGGTGGTT-3′MS2 Set 3 F: 5′-TGAACTTACACCAGCCCAGCACTT-3′ R:5′-TTCCGGCACCAGGTTAATGGTGGTTT-3′ Den-universal F:5-TCAATATGCTAAAACGCGCGAGAAACCG-3′ DEN-1 R: 5′-CGTCTCAGTGATCCGGGGG-3′DEN-2 R: 5′-CGCCACAAGGGCCATGAACAG-3′ DEN-3 R: 5′-TAACATCATCATGAGACAGAGC-3′ DEN-4 R: 5′-TGTTGTCTTAAACAAGAGAGGTC-3′β-actin F: 5′-TGACGGGGTCACCCACACTGTGCCCATCTA- 3′ R:5′-CTAGAAGCATTTGCGGTGGACGATGGAGGG- 3′

References for Example 9

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While the methods and articles described herein have been described withreference to specific embodiments, this application is intended to coverthose various changes and substitutions that can be made by those ofordinary skill in the art.

REFERENCES

The following publications, as well as all others referenced in thedisclosure, are incorporated herein by reference in their entirety:

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1-26. (canceled)
 27. A substrate comprising a surface onto which a DNAprobe is affixed, wherein the DNA probe comprises a sequencecomplementary to a unique sequence of a target molecule sequence thatincludes a recognition sequence for a nicking endonuclease.
 28. Thesubstrate of claim 27, wherein the substrate is a plastic or glass bead.29. The substrate of claim 27, wherein the substrate is a platecomprising a plurality of wells, the plurality of the wells comprisingone or more different DNA probes, each different probe comprising asequence complementary to a unique sequence of a target moleculesequence that includes a recognition sequence for a nickingendonuclease.
 30. The substrate of claim 27, wherein the probe comprisesa fluorescent tag that is released from the substrate surface if theprobe is cleaved by a nicking endonuclease.
 31. A kit comprising aplurality of desiccated substrates according to claim 27, differentsubstrates comprising one or more different probes.
 32. A method fordetecting the presence of a target nucleotide sequence in a sample ofDNA, the method comprising: (a) exposing a test sample comprising singlestranded DNA to a nicking endonuclease and a substrate surface accordingto claim 27 under conditions that would permit sequence-specifichybridization of the probe to a complementary target sequence, whereinthe probe comprises a sequence complementary to the target sequence thatalso includes a recognition sequence for the nicking endonuclease; and,(b) observing whether the probe is cleaved by the nicking endonuclease,wherein the presence of probe cleaved by the nicking endonucleaseindicates the presence of the target nucleotide sequence in the sampleDNA.
 33. The method of claim 32, wherein the substrate surface ontowhich the DNA probe is affixed comprises a surface of a plastic or glassbead.
 34. The method of claim 32, wherein the substrate surface ontowhich the DNA probe is affixed comprises a surface of a well in a plate.35. The method of claim 32, wherein the probe comprises a fluorescenttag that is released from the substrate surface if the probe is cleavedby the nicking endonuclease and the step of observing whether the probeis cleaved by the nicking endonuclease comprises detecting the presenceof fluorescent tag released from the substrate surface.
 36. The methodof claim 32, wherein prior to exposing the test sample comprising singlestranded DNA to the nicking endonuclease and the substrate surface ontowhich the DNA probe is affixed, the substrate surface onto which the DNAprobe is affixed is desiccated.
 37. The method of claim 32, whereinexposing the test sample comprising single stranded DNA to the nickingendonuclease and the substrate surface onto which a DNA probe is affixedcomprises exposing the test sample to a plurality of different beads,each comprising a substrate surface, each different substrate surfacecomprising a different DNA probe.
 38. A DNA probe comprising a sequencecomplementary to a unique sequence of a target DNA molecule that alsoincludes a recognition sequence for a nicking endonuclease, afluorescent tag, and a fluorescence quencher, the tag and quencher beinglocated on different sides of the recognition sequence for the nickingendonuclease, a first stem portion of the probe being capable ofhybridizing to a second stem portion of the probe unless the probe iscleaved at a cut site of the nicking endonuclease, the first and secondstem portions being separated by a loop portion, the tag and quencherbeing located in the probe such that the quencher is effective to quenchfluorescent emissions of the tag when the stem portions are hybridizedto each other.
 39. The probe of claim 38, wherein the recognitionsequence for the nicking endonuclease is located in the loop portion.40. The probe of claim 38, wherein the recognition sequence for thenicking endonuclease is located in one stem portion and the other stemportion includes a mismatch so that the probe does not comprise a duplexrecognition sequence for the nicking endonuclease.
 41. A method fordetecting the presence of a target nucleotide sequence in a sample ofDNA; the method comprising: (a) exposing a test sample comprising singlestranded DNA to a DNA probe according to claim 38 and a nickingendonuclease under conditions that would permit sequence-specifichybridization of the probe to a complementary target sequence, whereinthe probe comprises a sequence complementary to the target sequence thatalso includes a recognition sequence for the nicking endonuclease, afluorescent tag, and a fluorescence quencher, the tag and quencher beingsituated on different sides of the recognition sequence for the nickingendonuclease, a first stern portion of the probe being capable ofhybridizing to a second stem portion of the probe, the first and secondstem portions being separated by a loop portion, the tag and quencherbeing located in the probe such that the quencher is effective to quenchfluorescent emissions of the tag when the stem portions are hybridizedto each other; and, (b) observing whether the probe is cleaved by thenicking endonuclease, wherein the presence of fluorescent emissions ofthe fluorescent tag indicates the presence of the target nucleotidesequence in the sample DNA.
 42. The method of claim 41, wherein therecognition sequence for the nicking endonuclease is located in the loopportion.
 43. The method of claim 41, wherein the recognition sequencefor the nicking endonuclease is located in one stem portion and theother stem portion includes a mismatch so that the probe does notcomprise a duplex recognition sequence for the nicking endonuclease. 44.A method for detecting the presence of an RNA pathogen genome andoptionally a DNA pathogen genome in a sample of biological materialwherein the sample comprises a plurality of unpurified contaminants, themethod comprising: (a) performing a reverse transcription procedurecapable of reverse transcribing an RNA molecule in said sample ofbiological material into a complementary DNA molecule, (b) performingmultiple displacement amplification to amplify DNA in the sample ofbiological material to form an amplified sample product; (c)simultaneously exposing all or part of the amplified sample product to aplurality of DNA probes directed to a plurality of different pathogensand/or pathogen strains, wherein each probe comprises a sequencecomplementary to a unique sequence known to be present in a transcriptof a pathogen genome or a unique sequence known to be present in a DNAgenome of a pathogen that also includes a recognition sequence for thenicking endonuclease; and, (d) observing whether the probe is cleaved bythe nicking endonuclease, wherein the presence of probe cleaved by thenicking endonuclease indicates the presence of a pathogen in the sampleof biological material.
 45. A kit for detection and/or identification ofa dengue virus comprising one or more NESA probes comprising sequencesselected from among: 5′-CGTACTAGGATCACAAGAAGGA-3′,5′-TTGGATCATAGGGTATTGGATCTA-3′, 5′-GTTGTCCTTGGATCGCAAGAGGGA-3′,5′-GTTGTCCTTGGATCGCAAGAGGGATT-3′, 5′-AGTGCTGGGATCTCAGGAAGGA-3′ and5′-AGTGCTGGGATCTCAGGAAGGATTTT-3′ . . .


46. A method for making one or more NESA probes for the detection of apathogen genome, the method comprising: (a) identifying nucleotide,sequences of about 16-25 nucleotides in a pathogen genome that include anicking endonuclease recognition sequence, (b) discarding identifiedsequences that are not unique to the pathogen genome to produce alisting of unique identified sequences, (c) synthesizing one or moreprobes comprising unique identified sequences; and, (d) contacting oneor more samples of biological material with said one or more synthesizedprobes and the nicking endonuclease in the presence of one or morecommon contaminants, thereby validating the synthesized probe or probes.